EP3535406A1 - Production of steviol glycosides in recombinant hosts - Google Patents

Production of steviol glycosides in recombinant hosts

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Publication number
EP3535406A1
EP3535406A1 EP17801379.3A EP17801379A EP3535406A1 EP 3535406 A1 EP3535406 A1 EP 3535406A1 EP 17801379 A EP17801379 A EP 17801379A EP 3535406 A1 EP3535406 A1 EP 3535406A1
Authority
EP
European Patent Office
Prior art keywords
steviol
polypeptide
glycosides
seq
glucose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP17801379.3A
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German (de)
French (fr)
Inventor
Jon HEAL
Philipp BERNINGER
Kim OLSSON
Joe SHERIDAN
Laura OCCHIPINTI
Laura BREMBATI
Christian NYFFENAGER
Christophe FOLLY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evolva Holding SA
Original Assignee
Evolva AG
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Publication date
Application filed by Evolva AG filed Critical Evolva AG
Publication of EP3535406A1 publication Critical patent/EP3535406A1/en
Withdrawn legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/44Preparation of O-glycosides, e.g. glucosides
    • C12P19/56Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30Artificial sweetening agents
    • A23L27/33Artificial sweetening agents containing sugars or derivatives
    • A23L27/36Terpene glycosides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52Genes encoding for enzymes or proenzymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1048Glycosyltransferases (2.4)
    • C12N9/1051Hexosyltransferases (2.4.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P15/00Preparation of compounds containing at least three condensed carbocyclic rings
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y204/00Glycosyltransferases (2.4)
    • C12Y204/01Hexosyltransferases (2.4.1)
    • C12Y204/01126Hydroxycinnamate 4-beta-glucosyltransferase (2.4.1.126)

Definitions

  • the polypeptide capable of synthesizing enf-kaurene comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:44, 46, 48, 50, or 52;
  • the recombinant host cell is a Saccharomyces cerevisiae cell.
  • the recombinant host cell is a Yarrowia lipolytica cell.
  • polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
  • the term "cell culture” refers to a culture medium comprising one or more recombinant hosts.
  • a cell culture may comprise a single strain of recombinant host, or may comprise two or more distinct host strains.
  • the culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., UDP-glucose, UDP-rhamnose, UDP- xylose, N-acetyl-glucosamine, glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), etc.
  • steviol glycoside refers to Rebaudioside A (RebA) (CAS # 58543-16-1 ), Rebaudioside B (RebB) (CAS # 58543-17-2), Rebaudioside C (RebC) (CAS # 63550-99-2), Rebaudioside D (RebD) (CAS # 63279-13-0), Rebaudioside E (RebE) (CAS # 63279-14-1 ), Rebaudioside F (RebF) (CAS # 438045-89-7), Rebaudioside M (RebM) (CAS # 1220616-44-3), rubusoside (CAS # 63849-39-4), Dulcoside A (CAS # 64432-06-0), Rebaudioside I (Rebl) (MassBank Record: FU000332), Rebaudioside Q (RebQ), 1 ,2-Stevioside (CAS # 57817-89-7), 1 ,3-Stevioside (RebG), Steviol-1 ,2-Bioside (MassBank Record: FU000
  • a culture broth can comprise i) approximately 0.02-0.03 g/L MnCI 2 dihydrate and approximately 0.5-3.8 g/L MgS0 4 heptahydrate, ii) approximately 0.03-0.06 g/L MnCI 2 dihydrate and approximately 0.5-3.8 g/L MgS0 4 heptahydrate, and/or iii) approximately 0.03-0.17 g/L MnCI 2 dihydrate and approximately 0.5-7.3 g/L MgS0 4 heptahydrate.
  • a culture broth can comprise one or more steviol glycosides produced by a recombinant host, as described herein.
  • a recombinant host comprises a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (SEQ ID NO:7), a nucleic acid encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (SEQ ID NO:9), a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (SEQ ID NO:4), a nucleic acid encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-
  • one or more steviol glycosides or glycosides of a steviol precursor is produced by whole cell bioconversion.
  • a host cell expressing one or more enzymes involved in the steviol glycoside pathway takes up and modifies a steviol glycoside precursor in the cell; following modification in vivo, a steviol glycoside remains in the cell and/or is excreted into the culture medium.
  • the cell is permeabilized to take up a substrate to be modified or to excrete a modified product.
  • a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out.
  • the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween.
  • the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB).
  • CTAB cetyltrimethylammonium bromide
  • the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock.
  • polypeptides suitable for producing steviol glycosides or glycosides of steviol precursors such as kaurenoate-19-O-glucoside (19-KMG), steviol-19-0- glucoside (19-SMG), and rubusoside, in vitro, in a recombinant host, or by whole cell bioconversion include a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, such as a functional homolog of UGT74G1 (SEQ ID NO:4).
  • valine at residue 1 1 1 a valine at residue 1 1 1
  • 1 13 e.g., a cysteine at residue 1 13
  • 1 15 e.g., a valine at residue 1 15
  • 1 19 e.g. , a phenylalanine at residue 1 19
  • 120 e.g., a leucine at residue 120
  • 121 e.g.
  • a proline at residue 121 a proline at residue 121 ); 123 (e.g., an alanine at residue 123); 128 (e.g., a lysine at residue 128); 129 (e.g., a glutamine at residue 129); 135 (e.g., an alanine at residue 135); 136 (e.g., an alanine at residue 136); 140 (e.g. , an asparagine at residue 140); 141 (e.g.
  • UGT74G1 variants having one substitution e.g., L15V, F18Y, M79A, E87D, G31 S, E83D, N51 K, E75D, T49I, D99E, S96T, C73F, S84A, A68T, Q67E, I16L, I28L, G31A, S377T, M1 19F, E456K, L181V, L385F, N183D, E176D, F209L, N21 1 H, V143A, R297W, A410E, L390W, N252Y, S212T, V232T, 11 15V, G329E, T224M, I295L, T328S, L409I, D387E, D449N, V123A, M373V, V285A, Q204K, S189A, D247E, G135A, 11 1 1V
  • inclusion of UGT74G1 variants as otherwise described herein in an in vitro reaction mixture also comprising en/-kaurenoic acid, steviol, and/or 13-SMG increases accumulation of rubusoside, 19-SMG, and/or 19-KMG relative to a reaction mixture comprising, e.g., a UGT74G1 polypeptide having the amino acid sequence set forth in SEQ ID NO:4.
  • expression of UGT74G1 variants that increase accumulation of rubusoside also results in increased accumulation of 19-SMG and 19-KMG.
  • expression of UGT74G1 variants that increase accumulation of rubusoside also results in increased accumulation of 19-SMG, but decreased accumulation of 19-KMG.
  • expression of a UGT74G1 variant having more than one substitution results in increased rubusoside.
  • a substitution e.g., A335S, G407E, and Q91 E; D99E, L322F, and S192D; C73F, S146N, and T380S; E83D, R426K, and Q91 E; D434E, T380S, and I389V; or G361A, V325E, and I187Y, results in increased rubusoside.
  • steviol glycosides can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization.
  • steviol glycosides can be isolated by methods not limited to ion exchange chromatography, reversed-phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon columns and/or decoloring steps.
  • a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
  • a functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs.
  • ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk clustalw).
  • a number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g. , gram-negative bacteria, yeast, and fungi.
  • a species and strain selected for use as a steviol glycoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
  • suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia (formally known as Hansuela), Scheffersomyces, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces, Humicola, Issatchenkia, Brettanomyces, Yamadazyma, Lachancea, Zygosaccharomyces, Komagataella, Kazachstania, Xanthophyllomyces, Geotrichum, Blakeslea, Dunaliella, Haematococcus, Chlorella, Und
  • Physcomitrella mosses when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
  • Geotrichum is a fungi commonly found in soil, water and sewage worldwide. It's often identified in plants, cereal and diary products. Species include, for example but are not limited to, G. candidum and G. klebahnii (see Carmichael et al. , Mycologica, 1957, 49(6):820-830).
  • Kazachstania is a yeast genus in the family Sacchromycetaceae.
  • a microorganism can be a cyanobacterial cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis.
  • Debaromyces e.g. , D. hansenuis and D. hansenii
  • Scheffersomyces e.g., S. stipis
  • Rhodosporidium e.g., R. toruloides
  • Pachysolen e.g., P.
  • the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the sweetening profile desired for a given food product and that have a proportion of each steviol glycoside that is consistent from batch to batch.
  • hosts described herein do not produce or produce a reduced amount of undesired plant by-products found in Stevia extracts.
  • steviol glycoside compositions produced by the recombinant hosts described herein are distinguishable from compositions derived from Stevia plants.
  • a recombinant microorganism can be grown in a mixed culture to produce steviol and/or steviol glycosides.
  • a first microorganism can comprise one or more biosynthesis genes for producing a steviol glycoside precursor
  • a second microorganism comprises steviol glycoside biosynthesis genes. The product produced by the second, or final microorganism is then recovered.
  • a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
  • UGT74G1 variants produce one or more steviol glycosides or glycosides of a steviol precursor in relative amounts different than those of a wild-type UGT74G1 polypeptide.
  • several of the variants of Table 4 produce rubusoside and 19-SMG in relative amounts different than those of a wild-type UGT74G1 polypeptide (see e.g. , Example 21 of WO 201 1/153378).
  • DNVKQWLFPE CFHYLLKTQA ADGSWGSLPT TQTAGILDTA SAVLALLCHA QEPLQILDVS 120

Abstract

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.

Description

PRODUCTION OF STEVIOL GLYCOSIDES IN RECOMBINANT HOSTS
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This disclosure relates to recombinant production of steviol glycosides, glycosides of steviol precursors, and steviol glycoside precursors in recombinant hosts. In particular, this disclosure relates to production of steviol glycosides comprising steviol-13-O-glucoside (13- SMG), steviol-19-O-glucoside (19-SMG), steviol-1 ,2-bioside, steviol-1 ,3-bioside, 1 ,2-stevioside, 1 ,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (Rebl), dulcoside A, mono- glycosylated en/-kaurenoic acids, di-glycosylated en/-kaurenoic acids, tri-glycosylated ent- kaurenoic acids, mono-glycosylated enf-kaurenols (e.g., kaurenoate-19-O-glucoside or 19- KMG), di-glycosylated enf-kaurenols, tri-glycosylated en/-kaurenols, tri-glycosylated steviol glycosides, tetra-glycosylated steviol glycosides, penta-glycosylated steviol glycosides, hexa- glycosylated steviol glycosides, hepta-glycosylated steviol glycosides, or isomers thereof in recombinant hosts.
Description of Related Art
[0002] Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries. The sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking. Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine, and sucralose. Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevia rebaudiana. Stevia is commonly grown in South America and Asia for commercial production of stevia extract. Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener.
[0003] Chemical structures for several steviol glycosides are shown in Figure 1 , including the diterpene steviol and various steviol glycosides. Extracts of the Stevia plant generally comprise steviol glycosides that contribute to the sweet flavor, although the amount of each steviol glycoside often varies, inter alia, among different production batches.
[0004] Recovery and purification of steviol glycosides from the Stevia plant have proven to be labor intensive and inefficient. Moreover, steviol glycoside compositions obtained from a plant-derived Stevia extract generally contain Stevia plant-derived components that can contribute to off-flavors. As such, there remains a need for a recombinant production system that can accumulate high yields of desired steviol glycosides, such as RebD and RebM and produce steviol glycoside compositions that are enriched for a one or more desired steviol glycosides relative to a steviol glycoside composition of Stevia plant with a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract. There also remains a need for improved production of steviol glycosides in recombinant hosts for commercial uses. As well, there remains a need for identifying enzymes selective towards particular substrates to produce one or more specific steviol glycosides. In some aspects, there remains a need to increase the catalytic capability of enzymes with 19-0 glycosylation activity in order to produce higher yields of steviol glycosides.
SUMMARY OF THE INVENTION
[0005] It is against the above background that the present invention provides certain advantages and advancements over the prior art.
[0006] Although this invention as disclosed herein is not limited to specific advantages or functionalities (such for example, the ability to scale up production of a one or more steviol glycosides or glycosides of a steviol precursor, purify the one or more steviol glycosides or glycosides of the steviol precursor, and produce steviol glycoside compositions where the different proportions of the various steviol glycosides provide the advantage of having a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract), the invention provides a recombinant host cell capable of producing one or more steviol glycosides or glycosides of a steviol precursor, comprising a recombinant gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 110, 1 1 1 , 1 13, 1 15, 119-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191 -195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-41 1 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4.
[0007] In one aspect of the recombinant host cells disclosed herein, the polypeptide comprises at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31 S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T1 10P, 11 1 1V, A1 13C, 11 15V, M1 19F, T120L, E121 P, V123A, I 128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I 156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N21 1 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S411 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
[0008] In one aspect, the recombinant host cells disclosed herein further comprise:
(a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);
(b) a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP;
(c) a gene encoding an a polypeptide capable of synthesizing en/-kaurene from ent- copalyl diphosphate;
(d) a gene encoding a polypeptide capable of synthesizing en/-kaurenoic acid from en/-kaurene; (e) a gene encoding a polypeptide capable of synthesizing steviol from ent- kaurenoic acid;
(f) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;
(g) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;
(h) a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; and/or
(i) a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
wherein at least one of the genes is a recombinant gene.
In one aspect of the recombinant host cells disclosed herein:
(a) the polypeptide capable of synthesizing GGPP comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, or 1 16;
(b) the polypeptide capable of synthesizing enf-copalyl diphosphate comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:34, 36, 38, 40, or 42;
(c) the polypeptide capable of synthesizing enf-kaurene comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:44, 46, 48, 50, or 52;
(d) the polypeptide capable of synthesizing enf-kaurenoic acid comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:60, 62, 66, 68, 70, 72, 74, 76, or 1 17;
(e) the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:78, 80, 82, 84, 86, 88, 90, or 92; (f) the polypeptide capable of synthesizing steviol comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:94, 97, 100, 101 , 102, 103, 104, 106, 108, 1 10, 1 12, or 114;
(g) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(h) the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(i) the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.
[0010] In one aspect of the recombinant host cells disclosed herein, the one or more steviol glycosides or glycosides of the steviol precursor comprises kaurenoate-19-O-glucoside (19- KMG), steviol-13-O-glucoside (13-SMG), steviol-1 ,2-Bioside, steviol-1 ,3-Bioside, steviol-19-O- glucoside (19-SMG), 1 ,2-Stevioside, 1 ,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (Rebl), dulcoside A, a mono-glycosylated enf-kaurenoic acid, a di-glycosylated eni-kaurenoic acid, a tri-glycosylated en/-kaurenoic acid, a mono-glycosylated enf-kaurenols, a di-glycosylated en/-kaurenol, a tri-glycosylated enf-kaurenol, a tri-glycosylated steviol glycoside, a tetra- glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and/or an isomer thereof.
[0011] In one aspect of the recombinant host cells disclosed herein, the expression of the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having the one or more amino acid substitutions, increases or decreases the amount of 19- KMG, 19-SMG, and/or rubusoside produced by the cell by at least about 5%, 10%, 25%, 50%, or 100% relative to a corresponding host expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group lacking the one or more amino acid substitutions.
[0012] In one aspect of the recombinant host cells disclosed herein, the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, an archaeal cell, or a bacterial cell.
[0013] In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a Saccharomyces cerevisiae cell.
[0014] In one aspect of the recombinant host cells disclosed herein, the recombinant host cell is a Yarrowia lipolytica cell.
[0015] The invention also provides a method of producing one or more steviol glycosides or glycosides of a steviol precursor in a cell culture, comprising culturing the recombinant host cells disclosed herein in the cell culture, under conditions in which the genes are expressed; and wherein the one or more steviol glycosides or glycosides of the steviol precursor is produced by the recombinant host cell.
[0016] In one aspect of the methods disclosed herein, the genes are constitutively expressed.
[0017] In one aspect of the methods disclosed herein, the expression of the genes is induced.
[0018] In one aspect, the methods disclosed herein further comprise isolating the produced one or more steviol glycosides or glycosides of the steviol precursor from the cell culture.
[0019] In one aspect of the methods disclosed herein, the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or glycosides of the steviol precursor, and:
(a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or (b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or
(c) crystallizing or extracting the produced one or more steviol glycosides or glycosides of the steviol precursor;
thereby isolating the produced one or more steviol glycosides or glycosides of the steviol precursor.
[0020] In one aspect, the methods disclosed herein further comprise recovering the one or more steviol glycosides or glycosides of the steviol precursor from the cell culture.
[0021] In one aspect of the methods disclosed herein, the recovered one or more steviol glycosides or glycosides of the steviol precursor is enriched for the one or more steviol glycosides or glycosides of the steviol precursor relative to a steviol glycoside composition of Stevia plant and has a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract.
[0022] The invention also provides a method for producing one or more steviol glycosides or glycosides of a steviol precursor, comprising whole-cell bioconversion of a plant-derived or synthetic steviol, steviol precursors, and/or steviol glycosides in a cell culture of a recombinant host cell using a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179- 181 , 183-189, 191-195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-411 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4; and, optionally, one or more of:
(a) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, comprising a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; (b) the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(c) the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;
wherein at least one of the polypeptide is a recombinant polypeptide; and
producing the one or more steviol glycosides or glycosides of the steviol precursor thereby.
[0023] In one aspect of the methods disclosed herein, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group comprises at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31 S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T1 10P, 11 11V, A113C, 11 15V, M1 19F, T120L, E121 P, V123A, I 128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N21 1 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S41 1 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V. [0024] In one aspect of the methods disclosed herein, the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, an archaeal cell or a bacterial cell.
[0025] In one aspect of the methods disclosed herein, the recombinant host cell is a Saccharomyces cerevisiae cell.
[0026] In one aspect of the methods disclosed herein, the recombinant host cell is a Yarrowia lipolytica cell.
[0027] The invention also provides an in vitro method for producing one or more steviol glycosides or glycosides of a steviol precursor comprising adding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 119-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191-195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-41 1 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4; and, optionally, one or more of:
(a) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, comprising a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(b) the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(c) the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;
and a plant-derived or synthetic steviol, steviol precursors, and/or steviol glycosides to a reaction mixture;
wherein at least one of the polypeptide is a recombinant polypeptide; and
producing the one or more steviol glycosides or glycosides of the steviol precursor thereby.
[0028] In one aspect of the methods disclosed herein, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group comprises at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31 S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T1 10P, 11 11V, A113C, 11 15V, M1 19F, T120L, E121 P, V123A, I 128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N21 1 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S41 1 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
[0029] In one aspect of the methods disclosed herein, the reaction mixture comprises:
(a) one or more steviol glycosides or glycosides of the steviol precursor;
(b) a UGT polypeptide;
(c) uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N- acetyl-glucosamine; and/or
(d) reaction buffer and/or salts. [0030] In one aspect of the methods disclosed herein, the one or more steviol glycosides or glycosides of the steviol precursor comprises 19-KMG, 13-SMG, steviol-1 ,2-Bioside, steviol-1 ,3- Bioside, 19-SMG, 1 ,2-Stevioside, RebG, rubusoside, RebA, RebB, RebC, RebD, RebE, RebF, RebM, RebQ, Rebl, dulcoside A, a mono-glycosylated en/-kaurenoic acid, a di-glycosylated ent- kaurenoic acid, a tri-glycosylated en/-kaurenoic acid, a mono-glycosylated enf-kaurenols, a di- glycosylated en/-kaurenol, a tri-glycosylated enf-kaurenol, a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and/or an isomer thereof.
[0031] The invention also provides a cell culture, comprising the recombinant host cells disclosed herein, the cell culture further comprising:
(a) the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, UDP-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids;
wherein the one or more steviol glycosides or glycosides of the steviol precursor are present at a concentration of at least 1 mg/liter of the cell culture;
wherein the cell culture is enriched for the one or more steviol glycosides or glycosides of the steviol precursor relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
[0032] The invention also provides a cell lysate from the recombinant host cells disclosed herein grown in the cell culture, comprising:
(a) the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, UDP-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base, YNB, and/or amino acids; wherein the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell is present at a concentration of at least 1 mg/liter of the cell culture.
[0033] The invention also provides one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cells disclosed herein; wherein the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant- derived Stevia extract.
[0034] The invention also provides one or more steviol glycosides or glycosides of the steviol precursor produced by the methods disclosed herein; wherein the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
[0035] The invention also provides sweetener compositions, comprising the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell or the methods disclosed herein.
[0036] The invention also provides a food product, comprising the sweetener compositions disclosed herein.
[0037] The invention also provides a beverage or a beverage concentrate, comprising the sweetener compositions disclosed herein.
[0038] The invention also provides an isolated nucleic acid molecule encoding a polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191-195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-411 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4.
[0039] The invention also provides an isolated nucleic acid molecule encoding a polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31 S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T1 10P, 11 1 1V, A1 13C, 11 15V, M119F, T120L, E121 P, V123A, I128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I 180F, L181V, N183D, H184P, E185G, Q186S, I 187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N211 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S41 1 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
[0040] In one aspect of the nucleic acids disclosed herein, the nucleic acid is cDNA.
[0041] The invention also provides a polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86 88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191 -195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-41 1 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4.
[0042] The invention also provides a polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31 S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T1 10P, 11 1 1V, A1 13C, 11 15V, M1 19F, T120L, E121 P, V123A, I 128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N21 1 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S41 1 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
[0043] In one aspect of the polypeptides or the catalytically active portions thereof, wherein the polypeptide or the catalytically active portion thereof is a purified polypeptide or a catalytically active portion thereof.
[0044] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
[0046] Figure 1 shows representative primary steviol glycoside glycosylation reactions catalyzed by suitable UGT enzymes and chemical structures for several steviol glycoside compounds.
[0047] Figure 2 shows the biochemical pathway for the production of steviol, glycosylated en/-kaurenoic acid, and glycosylated enf-kaurenol from prenyl phosphates.
[0048] Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0049] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
[0050] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.
[0051] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
[0052] For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
[0053] Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et a/., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et a/., 1990, Academic Press, San Diego, CA).
[0054] As used herein, the terms "polynucleotide," "nucleotide," "oligonucleotide," and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
[0055] As used herein, the terms "microorganism," "microorganism host," "microorganism host cell," "recombinant host," and "recombinant host cell" can be used interchangeably. As used herein, the term "recombinant host" is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein ("expressed"), and other genes or DNA sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g. , to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms.
[0056] As used herein, the term "cell culture" refers to a culture medium comprising one or more recombinant hosts. A cell culture may comprise a single strain of recombinant host, or may comprise two or more distinct host strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., UDP-glucose, UDP-rhamnose, UDP- xylose, N-acetyl-glucosamine, glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), etc.
[0057] As used herein, the term "recombinant gene" refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. "Introduced," or "augmented" in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. In some aspects, said recombinant genes are encoded by cDNA. In other embodiments, recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
[0058] As used herein, the term "engineered biosynthetic pathway" refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
[0059] As used herein, the term "endogenous" gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some embodiments, the endogenous gene is a yeast gene. In some embodiments, the gene is endogenous to S. cerevisiae, including, but not limited to S. cerevisiae strain S288C. In some embodiments, an endogenous yeast gene is overexpressed. As used herein, the term "overexpress" is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism. See, e.g., Prelich, 2012, Genetics 190:841 -54. In some embodiments, an endogenous yeast gene is deleted. See, e.g. , Giaever & Nislow, 2014, Genetics 197(2) :451 -65. As used herein, the terms "deletion," "deleted," "knockout," and "knocked out" can be used interchangeably to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism, including, but not limited to, S. cerevisiae.
[0060] As used herein, the terms "heterologous sequence" and "heterologous coding sequence" are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
[0061] As used herein, the terms "heterologous sequence" and "heterologous coding sequence" are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
[0062] As used herein, the term "constitutive," "constitutive expression," or "constitutively expressed" refers to a continuous transcription of a gene resulting in the continuous expression of a protein.
[0063] As used herein, the term "inducible," "inducible expression," or "inducibly expressed" refers to the expression of a gene in response to a stumuli. Stimuli include, but are not limited to, chemicals, stress, or biotic stimuli.
[0064] A "selectable marker" can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change. Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art (see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, PCR or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g. , Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev. Genetics 36:153-173 and U.S. 2006/0014264). Alternatively, a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
[0065] As used herein, the terms "variant" and "mutant" are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild-type sequence of a particular protein.
[0066] As used herein, the term "inactive fragment" is a fragment of the gene that encodes a protein having, e.g., less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene. Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence. This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with inactivation thereof.
[0067] As used herein, the term "steviol glycoside" refers to Rebaudioside A (RebA) (CAS # 58543-16-1 ), Rebaudioside B (RebB) (CAS # 58543-17-2), Rebaudioside C (RebC) (CAS # 63550-99-2), Rebaudioside D (RebD) (CAS # 63279-13-0), Rebaudioside E (RebE) (CAS # 63279-14-1 ), Rebaudioside F (RebF) (CAS # 438045-89-7), Rebaudioside M (RebM) (CAS # 1220616-44-3), rubusoside (CAS # 63849-39-4), Dulcoside A (CAS # 64432-06-0), Rebaudioside I (Rebl) (MassBank Record: FU000332), Rebaudioside Q (RebQ), 1 ,2-Stevioside (CAS # 57817-89-7), 1 ,3-Stevioside (RebG), Steviol-1 ,2-Bioside (MassBank Record: FU000299), Steviol-1 ,3-Bioside, Steviol-13-O-glucoside (13-SMG), Steviol-19-O-glucoside (19- SMG), a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta- glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and isomers thereof. See Figure 1 ; see also, Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org.
[0068] As used herein, the terms "steviol glycoside precursor" and "steviol glycoside precursor compound" are used to refer to intermediate compounds in the steviol glycoside biosynthetic pathway. Steviol glycoside precursors include, but are not limited to, geranylgeranyl diphosphate (GGPP), enf-copalyl-diphosphate, en/-kaurene, enf-kaurenol, ent- kaurenal, enf-kaurenoic acid, and steviol. See Figure 2. Also as used herein, the terms "steviol precursor" and "steviol precursor compound" are used to refer to intermediate compounds in the steviol biosynthetic pathway. Steviol precursors may also be steviol glycoside precursors, and include, but are not limited to, geranylgeranyl diphosphate (GGPP), enf-copalyl-diphosphate, en/-kaurene, enf-kaurenol, enf-kaurenal, and en/-kaurenoic acid. Also as used herein, the terms "steviol precursor" and "steviol precursor compound" are used to refer to intermediate compounds in the steviol biosynthetic pathway.
[0069] Also as used herein, the term "glycosides of a steviol precursor" is used to refer to steviol precursors that can be glycosylated, e.g. , tri-glycosylated en/-kaurenoic acid (ent- kaurenoic acid+3Glc), di-glycosylated en/-kaurenoic acid, mono-glycosylated eni-kaurenoic acid, tri-glycosylated enf-kaurenol, di-glycosylated enf-kaurenol (en/-kaurenol+2Glc), or mono- glycosylated ent-kaurenol (en/-kaurenol+1 Glc; e.g., kaurenoate-19-O-glucoside or 19-KMG). In some embodiments, steviol glycoside precursors are themselves steviol glycoside compounds. For example, 19-SMG, rubusoside, stevioside, and RebE are steviol glycoside precursors of RebM. See Figure 1 .
[0070] As used herein, the term "contact" is used to refer to any physical interaction between two objects. For example, the term "contact" may refer to the interaction between an enzyme and a substrate. In another example, the term "contact" may refer to the interaction between a liquid (e.g., a supernatant) and an adsorbent resin.
[0071] Steviol precursors may also be steviol glycoside precursors, and include, but are not limited to, geranylgeranyl diphosphate (GGPP), enf-copalyl-diphosphate, en/-kaurene, ent- kaurenol, enf-kaurenal, and enf-kaurenoic acid. Steviol glycosides and/or steviol glycoside precursors, or glycosides of a steviol precursor can be produced in vivo (i.e., in a recombinant host), in vitro (i.e. , enzymatically), or by whole cell bioconversion.
[0072] As used herein, the terms "produce" and "accumulate" can be used interchangeably to describe synthesis of steviol glycosides and steviol glycoside precursors in vivo, in vitro, or by whole cell bioconversion.
[0073] As used herein, the terms "culture broth," "culture medium," and "growth medium" can be used interchangeably to refer to a liquid or solid that supports growth of a cell. A culture broth can comprise glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids. The trace metals can be divalent cations, including, but not limited to, Mn2+ and/or Mg2+. In some embodiments, Mn2+ can be in the form of MnCI2 dihydrate and range from approximately 0.01 g/L to 100 g/L. In some embodiments, Mg can be in the form of MgS04 heptahydrate and range from approximately 0.01 g/L to 100 g/L. For example, a culture broth can comprise i) approximately 0.02-0.03 g/L MnCI2 dihydrate and approximately 0.5-3.8 g/L MgS04 heptahydrate, ii) approximately 0.03-0.06 g/L MnCI2 dihydrate and approximately 0.5-3.8 g/L MgS04 heptahydrate, and/or iii) approximately 0.03-0.17 g/L MnCI2 dihydrate and approximately 0.5-7.3 g/L MgS04 heptahydrate. Additionally, a culture broth can comprise one or more steviol glycosides produced by a recombinant host, as described herein.
[0074] Recombinant steviol glycoside-producing Saccharomyces cerevisiae (S. cerevisiae) strains are described in WO 201 1/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in their entirety. Methods of producing steviol glycosides in recombinant hosts, by whole cell bio-conversion, and in vitro are also described in WO 201 1/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
[0075] In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) (e.g. , a geranylgeranyl diphosphate synthase (GGPPS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent- copalyl diphosphate from GGPP (e.g. , a ent-copalyl diphosphate synthase (CDPS) polypeptide); a gene encoding a polypeptide capable of synthesizing en/-kaurene from eni-copalyl diphosphate (e.g., a kaurene synthase (KS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from eni-kaurene (e.g., a kaurene oxidase (KO) polypeptide); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a cytochrome P450 reductase (CPR) polypeptide or a P450 oxidoreductase (POR) polypeptide; for example, but not limited to a polypeptide capable of capable of reducing cytochrome P450 complex (e.g. , an electron transfer from NADPH to cytochrome P450 complex during conversion of NADPH to NADP+), which is utilized as a cofactor for terpenoid biosynthesis); a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid (e.g. , a steviol synthase (KAH) polypeptide); and/or a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., an ent-copalyl diphosphate synthase (CDPS) - en/-kaurene synthase (KS) polypeptide) can produce steviol in vivo. See, e.g. , Figure 1. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host. [0076] In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a UGT85C2 polypeptide); a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (e.g., a UGT76G1 polypeptide); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (e.g. , a UGT74G1 polypeptide); and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a UGT91 D2 or a EUGT1 1 polypeptide) can produce a steviol glycoside in vivo. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[0077] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host. For example, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing en/-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing eni-copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can produce a steviol glycoside and/or steviol glycoside precursors in vivo. See, e.g., Figures 1 and 2. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host. [0078] In some embodiments, a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
[0079] In some embodiments, a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
[0080] In some aspects, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, and/or a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, transfers a glucose molecule from uridine diphosphate glucose (UDP-glucose) to steviol and/or a steviol glycoside.
[0081] In some aspects, the polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 19), SEQ ID NO:22 (encoded by the nucleotide sequence set forth in SEQ ID NO:21), SEQ ID NO:24 (encoded by the nucleotide sequence set forth in SEQ ID NO:23), SEQ ID NO:26 (encoded by the nucleotide sequence set forth in SEQ ID NO:25), SEQ ID NO:28 (encoded by the nucleotide sequence set forth in SEQ ID NO:27), SEQ ID NO:30 (encoded by the nucleotide sequence set forth in SEQ ID NO:29), SEQ ID NO:32 (encoded by the nucleotide sequence set forth in SEQ ID NO:31), or SEQ ID NO: 1 16 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 1 15). [0082] In some aspects, the polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), SEQ ID NO:36 (encoded by the nucleotide sequence set forth in SEQ ID NO:35), SEQ ID NO:38 (encoded by the nucleotide sequence set forth in SEQ ID NO:37), SEQ ID NO:40 (encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (encoded by the nucleotide sequence set forth in SEQ ID NO:41). In some embodiments, the polypeptide capable of synthesizing ent- copalyl diphosphate from GGPP lacks a chloroplast transit peptide. For example, the polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP lacking a chloroplast transit polypeptide can comprise a polypeptide having an amino acid sequence set forth in SEQ ID NO:120 (encoded by the nucleotide sequence set forth in SEQ ID NO: 119).
[0083] In some aspects, the polypeptide capable of synthesizing en/-kaurene from ent- copalyl pyrophosphate comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:44 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43), SEQ ID NO:46 (encoded by the nucleotide sequence set forth in SEQ ID NO:45), SEQ ID NO:48 (encoded by the nucleotide sequence set forth in SEQ ID NO:47), SEQ ID NO:50 (encoded by the nucleotide sequence set forth in SEQ ID NO:49), or SEQ ID NO:52 (encoded by the nucleotide sequence set forth in SEQ ID NO:51).
[0084] In some aspects, the polypeptide capable of synthesizing en/-kaurenoic acid from en/-kaurene comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:60 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:59), SEQ ID NO:62 (encoded by the nucleotide sequence set forth in SEQ ID NO:61), SEQ ID NO: 1 17 (encoded by the nucleotide sequence set forth in SEQ ID NO:63 or SEQ ID NO:64), SEQ ID NO:66 (encoded by the nucleotide sequence set forth in SEQ ID NO:65), SEQ ID NO:68 (encoded by the nucleotide sequence set forth in SEQ ID NO:67), SEQ ID NO:70 (encoded by the nucleotide sequence set forth in SEQ ID NO:69), SEQ ID NO:72 (encoded by the nucleotide sequence set forth in SEQ ID NO:71), SEQ ID NO:74 (encoded by the nucleotide sequence set forth in SEQ ID NO:73), or SEQ ID NO:76 (encoded by the nucleotide sequence set forth in SEQ ID NO:75).
[0085] In some aspects, the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:78 (which can be encoded by the nucleotide sequence set forth in SEQ I D NO:77), SEQ ID NO:80 (encoded by the nucleotide sequence set forth in SEQ ID NO:79), SEQ ID NO:82 (encoded by the nucleotide sequence set forth in SEQ ID NO:81), SEQ ID NO:84 (encoded by the nucleotide sequence set forth in SEQ ID NO:83), SEQ ID NO:86 (encoded by the nucleotide sequence set forth in SEQ ID NO:85), SEQ ID NO:88 (encoded by the nucleotide sequence set forth in SEQ ID NO:87), SEQ ID NO:90 (encoded by the nucleotide sequence set forth in SEQ ID NO:89), or SEQ ID NO:92 (encoded by the nucleotide sequence set forth in SEQ ID NO:91).
[0086] In some aspects, the polypeptide capable of synthesizing steviol from eni-kaurenoic acid comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:94 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:93), SEQ ID NO:97 (encoded by the nucleotide sequence set forth in SEQ ID NO:95 or SEQ ID NO:96), SEQ ID NO: 100 (encoded by the nucleotide sequence set forth in SEQ ID NO:98 or SEQ ID NO:99), SEQ ID NO: 101 , SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 106 (encoded by the nucleotide sequence set forth in SEQ ID NO:105), SEQ ID NO:108 (encoded by the nucleotide sequence set forth in SEQ ID NO: 107), SEQ ID NO:1 10 (encoded by the nucleotide sequence set forth in SEQ ID NO: 109), SEQ ID NO: 1 12 (encoded by the nucleotide sequence set forth in SEQ ID NO:1 1 1), or SEQ ID NO: 1 14 (encoded by the nucleotide sequence set forth in SEQ ID NO:1 13).
[0087] In some embodiments, a recombinant host comprises a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (SEQ ID NO:7), a nucleic acid encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (SEQ ID NO:9), a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (SEQ ID NO:4), a nucleic acid encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (SEQ ID NO:16, SEQ ID NO: 1 1 , SEQ ID NO: 13). In some aspects, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6, the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:8, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group is encoded by the nucleotide sequence set forth in SEQ ID NO: 118 or SEQ ID NO:3, the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs:10, 12, 14, or 15.
[0088] In certain embodiments, the steviol glycoside produced is RebA, RebB, RebD, and/or RebM. RebA can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. RebB can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. RebD can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. RebM can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (see Figure 2).
[0089] In some embodiments, one or more steviol glycosides or glycosides of a steviol precursor is produced by whole cell bioconversion. For whole cell bioconversion to occur, a host cell expressing one or more enzymes involved in the steviol glycoside pathway takes up and modifies a steviol glycoside precursor in the cell; following modification in vivo, a steviol glycoside remains in the cell and/or is excreted into the culture medium. For example, a host cell expressing a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can take up steviol and glycosylate steviol in the cell; following glycosylation in vivo, a steviol glycoside can be excreted into the culture medium.
[0090] In some embodiments, the method of producing one or more steviol glycosides or glycosides of a steviol precursor disclosed herein comprises whole-cell bioconversion of plant- derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179- 181 , 183-189, 191-195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-411 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4; wherein the polypeptides is a recombinant polypeptide; and synthesizing the one or more steviol glycosides or the steviol glycoside composition thereby.
[0091] In some embodiments of the methods of producing one or more steviol glycosides or glycosides of a steviol precursor disclosed herein comprises whole-cell bioconversion of plant- derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell disclosed herein, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group comprises at least one amino acid substitution of SEQ ID NO:4 that is L15V, I 16L, F18Y, L20A, F27M, I28L, F30L, G31 S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T110P, 11 1 1V, A1 13C, 11 15V, M1 19F, T120L, E121 P, V123A, I128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N21 1 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S41 1 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
[0092] In some embodiments, the cell is permeabilized to take up a substrate to be modified or to excrete a modified product. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. In some embodiments, the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween. In some embodiments, the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB). In some embodiments, the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
[0093] In some embodiments, steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides are produced by co-culturing of two or more hosts. In some embodiments, one or more hosts, each expressing one or more enzymes involved in the steviol glycoside pathway, produce steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides. For example, a host expressing a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from en/-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate and a host expressing a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-0- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, produce one or more steviol glycosides.
[0094] In some embodiments, the steviol glycoside comprises, for example, but not limited to, steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), steviol-1 ,2-bioside, steviol-1 ,3-bioside, 1 ,2-stevioside, 1 ,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (Rebl), dulcoside A, mono-glycosylated en/-kaurenoic acids, di-glycosylated ent- kaurenoic acids, tri-glycosylated en/-kaurenoic acids, mono-glycosylated enf-kaurenols (e.g., kaurenoate-19-O-glucoside or 19-KMG), di-glycosylated enf-kaurenols, tri-glycosylated ent- kaurenols, tri-glycosylated steviol glycosides, tetra-glycosylated steviol glycosides, penta- glycosylated steviol glycosides, hexa-glycosylated steviol glycosides, hepta-glycosylated steviol glycosides, or isomers thereof.
[0095] In some embodiments, polypeptides suitable for producing steviol glycosides or glycosides of steviol precursors, such as kaurenoate-19-O-glucoside (19-KMG), steviol-19-0- glucoside (19-SMG), and rubusoside, in vitro, in a recombinant host, or by whole cell bioconversion include a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, such as a functional homolog of UGT74G1 (SEQ ID NO:4). As described in section "Functional Homologs" below, functional homologs disclosed herein can include, for example but not limited to, conservative amino acid substitutions, such as, for example, substitution of one hydrophobic residue for another or substitution of one polar residue for another.
[0096] In some embodiments, a useful UGT74G1 homolog can have one or more amino acid substitutions at residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191-195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-41 1 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458. See, Table 1 .
[0097] Non-limiting examples of useful UGT74G1 homologs include polypeptides having substitutions (with respect to SEQ I D NO:4) at residue 15 (e.g., a valine at residue 15); 16 (e.g., a leucine at reside 16); 18 (e.g. , a tyrosine at residue 18); 20 (e.g., an alanine at residue 20); 27 (e.g., a methionine at residue 27); 28 (e.g., a leucine at residue 28); 30 (e.g. , a leucine at residue 30); 31 (e.g. , a serine or an alanine at residue 31); 49 (e.g. , an isoleucine at residue 49); 51 (e.g. , a lysine at residue 51 ); 67 (e.g. , a glutamic acid at residue 67); 68 (e.g., a threonine at residue 68); 73 (e.g., a phenylalanine at residue 73); 75 (e.g., an aspartic acid at residue 75); 79 (e.g., an alanine at residue 79); 81 (e.g., a tryptophan at residue 81 ); 83 (e.g., an aspartic acid or a lysine at residue 83); 84 (e.g., an alanine at residue 84); 86 (e.g. , an isoleucine at residue 86); 87 (e.g. , an aspartic acid at residue 87); 88 (e.g. , an arginine at residue 88); 90 (e.g. , a tryptophan at residue 90); 91 (e.g., a glutamic acid at residue 91 ); 96 (e.g. , a threonine at residue 96); 99 (e.g. , a glutamic acid at residue 99); 107 (e.g., a serine at residue 107); 1 10 (e.g., a proline at residue 1 10); 1 1 1 (e.g. , a valine at residue 1 1 1 ); 1 13 (e.g., a cysteine at residue 1 13); 1 15 (e.g., a valine at residue 1 15); 1 19 (e.g. , a phenylalanine at residue 1 19); 120 (e.g., a leucine at residue 120); 121 (e.g. , a proline at residue 121 ); 123 (e.g., an alanine at residue 123); 128 (e.g., a lysine at residue 128); 129 (e.g., a glutamine at residue 129); 135 (e.g., an alanine at residue 135); 136 (e.g., an alanine at residue 136); 140 (e.g. , an asparagine at residue 140); 141 (e.g. , a serine at residue 141 ); 143 (e.g., an alanine at residue 143); 146 (e.g., an asparagine at residue 146); 147 (e.g., an isoleucine at residue 147); 156 (e.g. , a leucine at residue 156); 162 (e.g., a threonine at residue 162); 166 (e.g. , a leucine at residue 166); 169 (e.g. , a leucine at residue 169); 173 (e.g. , a glutamic acid at residue 173); 176 (e.g., an aspartic acid at residue 176); 179 (e.g. , a serine at residue 179); 180 (e.g., a phenylalanine at residue 180); 181 (e.g. , a valine at residue 181); 183 (e.g. , an aspartic acid at residue 183); 184 (e.g. , a proline at residue 184); 185 (e.g. , a glycine at residue 185); 186 (e.g. , a serine at residue 186); 187 (e.g. , a tyrosine at residue 187); 188 (e.g., a proline at residue 188); 189 (e.g., an alanine at residue 189); 191 (e.g., a phenylalanine at residue 191 ); 192 (e.g., an aspartic acid at residue 192); 193 (e.g. , a methionine at residue 193); 194 (e.g., a valine at residue 194); 195 (e.g. , a valine at residue 195); 200 (e.g. , a serine at residue 200); 204 (e.g., a lysine at residue 204); 209 (e.g., a leucine at residue 209); 21 1 (e.g., a histidine at residue 21 1 ); 212 (e.g., a threonine at residue 212); 215 (e.g., a glutamic acid at residue 215); 221 (e.g. , a valine at residue 221); 222 (e.g. , an aspartic acid at residue 222); 224 (e.g., a methionine at residue 224); 232 (e.g. , a threonine at residue 232); 237 (e.g., an isoleucine at residue 237); 247 (e.g., a glutamic acid at residue 247); 252 (e.g. , a tyrosine at residue 252); 255 (e.g., a serine at residue 255); 257 (e.g. , a phenylalanine at residue 257); 259 (e.g., a proline at residue 259); 263 (e.g., an alanine at residue 263); 265 (e.g., an isoleucine at residue 265); 266 (e.g. , a lysine or a glutamic acid at residue 266); 269 (e.g., an asparagine at residue 269); 274 (e.g., a glycine at residue 274); 280 (e.g., a serine at residue 280); 284 (e.g. , a methionine at residue 284); 285 (e.g., an alanine at residue 285); 287 (e.g. , a leucine at residue 287); 292 (e.g., a methionine at residue 292); 295 (e.g. , a leucine or a methionine at residue 295); 296 (e.g., an alanine at residue 296); 297 (e.g., a tryptophan at residue 297); 298 (e.g., a glycine at residue 298); 300 (e.g., a lysine at residue 300); 301 (e.g. , an asparagine at residue 301); 303 (e.g., an asparagine at residue 303); 310 (e.g. , a valine at residue 310); 31 1 (e.g., an arginine at residue 31 1); 313 (e.g. , a serine at residue 313); 315 (e.g. , a glutamine at residue 315); 316 (e.g., an alanine at residue 316); 320 (e.g. , a lysine at residue 320); 322 (e.g. , a phenylalanine at residue 322); 325 (e.g. , a glutamic acid at residue 325); 326 (e.g., a threonine at residue 326); 328 (e.g., a serine at residue 328); 329 (e.g., a glutamic acid at residue 329); 332 (e.g. , isoleucine at residue 332); 333 (e.g. , a valine at residue 333); 335 (e.g. , a serine at residue 335); 338 (e.g., a proline at residue 338); 341 (e.g., a glutamic acid at residue 341); 346 (e.g., a lysine or a proline at residue 346); 347 (e.g., an alanine at residue 347); 357 (e.g. , tryptophan at residue 357); 364 (e.g., a leucine at residue 364); 370 (e.g., a methionine at residue 370); 371 (e.g., an isoleucine at residue 371); 373 (e.g., a valine at residue 373); 375 (e.g. , a leucine at residue 375); 376 (e.g., a tryptophan at residue 376); 377 (e.g., a threonine at residue 377); 380 (e.g., a serine at residue 380); 385 (e.g., a phenylalanine at residue 385); 387 (e.g., a glutamic acid at residue 387); 388 (e.g. , an aspartic acid at residue 388); 389 (e.g. , a valine at residue 389); 390 (e.g., a tryptophan at residue 390); 391 (e.g., a lysine at residue 391); 396 (e.g., an alanine at residue 396); 401 (e.g. , a lysine at residue 401 ); 407 (e.g., a glutamic acid at residue 407); 408 (e.g., a glutamic acid at residue 408); 409 (e.g. , an isoleucine at residue 409); 410 (e.g., a glutamic acid at residue 410); 41 1 (e.g., an aspartic acid at residue 41 1); 415 (e.g. , a glutamic acid at residue 415); 416 (e.g. , a valine at residue 416); 419 (e.g. , a glycine at residue 419); 424 (e.g. , a glutamic acid at residue 424); 426 (e.g., a lysine at residue 426); 427 (e.g. , a glutamic acid at residue 427); 434 (e.g., a glutamic acid at residue 434); 448 (e.g., a lysine at residue 448); 449 (e.g., an asparagine at residue 449); 455 (e.g. , an alanine at residue 455); 456 (e.g., a lysine at residue 456); or 458 (e.g., a valine at residue 458).
[0098] In some embodiments, UGT74G1 variants having one substitution (with respect to SEQ ID NO:4), e.g., L15V, F18Y, M79A, E87D, G31 S, E83D, N51 K, E75D, T49I, D99E, S96T, C73F, S84A, A68T, Q67E, I16L, I28L, G31A, S377T, M1 19F, E456K, L181V, L385F, N183D, E176D, F209L, N21 1 H, V143A, R297W, A410E, L390W, N252Y, S212T, V232T, 11 15V, G329E, T224M, I295L, T328S, L409I, D387E, D449N, V123A, M373V, V285A, Q204K, S189A, D247E, G135A, 11 1 1V, T120L, G316A, Q173E, V166L, 1221V, L147I, F376W, L284M, E162T, Q375L, S136A, E315Q, I333V, M265I, A141 S, E107S, E185G, V396A, L237I, Q186S, E320K, A200S, L195V, Q188P, Y257F, D269N, D341 E, D434E, K313S, L179S, S455A, E263A, K31 1 R, A259P, T1 10P, V292M, I326T, T296A, E222D, G391 K, K215E, 1310V, I156L, D303N, E121 P, V370M, K427E, I180F, E274G, I458V, A335S, S41 1 D, or F169L, accumulate rubusoside, 19-SMG, and/or 19-KMG.
[0099] In some embodiments, UGT74G1 variants having two substitutions (with respect to SEQ ID NO:4), e.g., E176D and F357W, accumulate rubusoside, 19-SMG, and/or 19-KMG. In some embodiments, UGT74G1 variants having three substitutions (with respect to SEQ ID NO:4), e.g. , F18Y, 1416V, and F27M; E87D, Q91 E, and I300K; E274G, L86I, and F30L; E83D, R426K, and Q91 E; S96T, V325E, and T88R; D99E, L322F, and S192D; C73F, S146N, and T380S; A259P, V371 I, and K90W; T49I, A280S, and A1 13C; V123A, M194V, and T88R; L181V, A280S, and L86I; N252Y, E129Q, and F30L; A68T, L322F, and A1 13C; S212T, F357W, and F30L; E75D, I300K, and F357W; A335S, G407E, and Q91 E; I16L, A113C, and M415E; G31 S, N255S, and I295M; S377T, E388D, and L86I; I180F, H184P, and E83K; Q188P, N408E, and E83K; K31 1 R, W191 F, and F27M; L195V, L20A, and E346P; M79A, V325E, and M415E; Q67E, N401 K, and D301 N; S84A, S347A, and I295M; A141 S, F27M, and V371 I; L179S, N266K, and E83K; Q186S, W191 F, and T88R; A410E, A298G, and L20A; K31 1 R, W191 F, and F27M; V285A, E388D, and L20A; E176D, L322F, and K90W; F169L, K90W, and N266E; E456K, N255S, and N401 K; V370M, N448K, and M194V; A200S, E129Q, and Q140N; L390W, N266K, and S192D; E320K, N266K, and M194V; M265I, I364L, and I187Y; E315Q, E129Q, and S192D; E222D, N408E, and Q140N; M1 19F, N255S, and A298G; D269N, R426K, and S146N; E185G, A298G, and Q193M; V232T, E388D, and Q140N; G316A, V325E, and I187Y; L409I, D301 N, and H184P; S189A, S146N, and I389V; V143A, 1416V, and H184P; S455A, Q193M, and E346K; R297W, S347A, and E346K; D341 E, Q193M, and L332I; D434E, T380S, and I389V; T328S, A280S, and L332I; Q375L, E419G, and R426K; 1221V, K338P, and I295M; T296A, I128K, and L332I; D449N, G407E, and H287L; T1 10P, K338P, and E346P; E121 P, 1416V, and H287L; L284M, I424E, and E346K; K427E, K338P, and I424E; D303N, N408E, and I300K; D247E, D301 N, and E346P; F376W, N448K, and I 128K; K313S, I 187Y, I 128K; G329E, G407E, and N266E; 11 1 1V, M415E, and H287L; G391 K, T380S, and I389V; V396A, N448K, and S347A; 1310V, V371 I, and N266E; Y257F, N401 K, and W191 F; S136A, I364L, and I424E; or V292M, E419G, and I364L, accumulate rubusoside, 19-SMG, and/or 19-KMG. See, Tables 2 and 3.
[00100] In some embodiments, expression of UGT74G1 variants as otherwise described herein in steviol glycoside producing S. cerevisiae strains (See, WO 2014/122227, which is hereby incorporated herein by reference in its entirety) increases accumulation of rubusoside, 19-SMG, and/or 19-KMG relative to steviol glycoside producing S. cerevisiae strains expressing, e.g., a UGT74G1 polypeptide having the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, inclusion of UGT74G1 variants as otherwise described herein in an in vitro reaction mixture also comprising en/-kaurenoic acid, steviol, and/or 13-SMG increases accumulation of rubusoside, 19-SMG, and/or 19-KMG relative to a reaction mixture comprising, e.g., a UGT74G1 polypeptide having the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, expression of UGT74G1 variants that increase accumulation of rubusoside also results in increased accumulation of 19-SMG and 19-KMG. In some embodiments, expression of UGT74G1 variants that increase accumulation of rubusoside also results in increased accumulation of 19-SMG, but decreased accumulation of 19-KMG. In some embodiments, expression of UGT74G1 variants that increase accumulation of rubusoside also results in increased accumulation of 19-KMG, but decreased accumulation of 19-SMG. In some embodiments, expression of UGT74G1 variants that increase accumulation of rubusoside also results in decreased accumulation of 19-SMG and 19-KMG.
[00101] In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-SMG also results in increased accumulation of rubusoside and 19-KMG. In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-SMG also results in increased accumulation of rubusoside, but decreased accumulation of 19-KMG. In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-SMG also results in increased accumulation of 19-KMG, but decreased accumulation of rubusoside. In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-SMG also results in decreased accumulation of rubusoside and 19-KMG. [00102] In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-KMG also results in increased accumulation of rubusoside and 19-SMG. In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-KMG also results in increased accumulation of rubusoside, but decreased accumulation of 19-SMG. In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-KMG also results in increased accumulation of 19-SMG, but decreased accumulation of rubusoside. In some embodiments, expression of UGT74G1 variants that increase accumulation of 19-KMG also results in decreased accumulation of rubusoside and 19-SMG.
[00103] In some embodiments, expression of a UGT74G1 variant having one substitution (with respect to SEQ ID NO:4), e.g. , N183D, D387E, L409I, G316A, T224M, V143A, A410E, L390W, S212T, Q204K, T120L, M79A, L237I, I295L, S136A, V285A, N21 1 H, V232T, or l_181V, results in increased rubusoside. In some embodiments, expression of a UGT74G1 variant having more than one substitution (with respect to SEQ ID NO:4), e.g., A335S, G407E, and Q91 E; D99E, L322F, and S192D; C73F, S146N, and T380S; E83D, R426K, and Q91 E; D434E, T380S, and I389V; or G361A, V325E, and I187Y, results in increased rubusoside.
[00104] In some embodiments, expression of a UGT74G1 variant having one substitution (with respect to SEQ ID NO:4), e.g., F169L, E176D, E456K, M1 19F, S377T, L15V, L385F, F18Y, L181V, N183D, F209L, V166L, R297W, C73F, D449N, E107S, N252Y, G135A, S189A, G31A, T49I, I180F, Q375L, Q186S, F376W, S41 1 D, V370M, 11 1 1V, 1221V, I458V, E87D, G31 S, V123A, K427E, L179S, I28L, I156L, S84A, I16L, D303N, E274G, Q188P, L284M, Q173E, 11 15V, V143A, 1310V, N21 1 H, E222D, E83D, A335S, V292M, D99E, T296A, E162T, E263A, A68T, S96T, A259P, A141 S, V285A, G329E, G391 K, K31 1 R, M79A, L195V, E320K, T1 10P, K215E, or I333V, results in increased 19-KMG. In some embodiments, expression of a UGT74G1 variant having more than one substitution (with respect to SEQ ID NO:4), e.g., V232T, E388D, and Q140N; E222D, N408E, and Q140N; S189A, S146N, and I389V; M1 19F, N255S, and A298G; A200S, E129Q, and Q140N; L181V, A280S, and L86I; S377T, E388D, and L86I; E456K, N255S, and N401 K; V123A, M194V, and T88R; C73F, S146N, and T380S; V370M, N448K, and M194V; D99E, L322F, and S192D; A259P, V371 I, and K90W; E315Q, E129Q, and S192D; M265I, I364L, and I187Y; T49I, A280S, and A113C; E320K, N266K, and M194V; L390W, N266K, and S192D; D269N, R426K, and S146N; E185G, A298G, and Q193M; S212T, F357W, and F30L; N252Y, E129Q, and F30L; V143A, 1416V, and H184P; L409I, D301 N, and H184P; R297W, S347A, and E346K; 116L, A1 13C, and M415E; A68T, L322F, and A1 13C; Q188P, N408E, and E83K; G31 S, N255S, and I295M; E75D, I300K, and F357W; G316A, V325E, and I187Y; S455A, Q193M, and E346K; E274G, L86I, and F30L; D341 E, Q193M, and L332I; E176D and F357W; I180F, H184P, and E83K; T328S, A280S, and L332I; D449N, G407E, and H287L; K313S, I187Y, and I128K; S96T, V325E, and T88R; V396A, N448K, and S347A; F18Y, 1416V, and F27M; 1221V, K338P, and I295M; K427E, K338P, and I424E; V292M, E419G, and I364L; G391 K, T380S, and I389V; G329E, G407E, and N266E; 1310V, V371 I, and N266E; M79A, V325E, and M415E; Q67E, N401 K, and D301 N; S84A, S347A, and I295M; L179S, N266K, and E83K; 11 1 1V, M415E, and H287L; Q186S, W191 F, and T88R; V285A, E388D, and L20A; F376W, N448K, and I128K; Y257F, N401 K, and W191 F; D247E, D301 N, and E346P; D303N, N408E, and I300K; A141 S, F27M, and V371 I; S136A, I364L, and I424E; K311 R, W191 F, and F27M; Q375L, E419G, and R426K; L195V, L20A, and E346P; A410E, A298G, and L20A; T1 10P, K338P, and E346P; E121 P, 1416V, and H287L; K31 1 R, W191 F, and F27M; E87D, Q91 E, and I300K; or L284M, I424E, and E346K, results in increased 19-KMG.
[00105] In some embodiments, expression of a UGT74G1 variant having one substitution (with respsect to SEQ ID NO:4), e.g., G316A, Q204K, S212T, A410E, I295L, T328S, T224M, L409I, D387E, T120L, S136A, A200S, M373V, E315Q, V143A, L390W, or M79A, results in increased 19-SMG. In some embodiments, expression of a UGT74G1 variant having more than one substitution (with respect to SEQ ID NO:4), e.g. , A335S, G407E, and Q91 E; T296A, I128K, and I332I; D434E, T380S, and I389V; or E87D, Q91 E, and I300K, results in increased 19-SMG.
[00106] In some embodiments, one or more steviol glycosides or glycosides of a steviol precursor produced in vivo, in vitro, or by whole cell bioconversion does not comprise or comprises a reduced amount or reduced level of plant-derived components than a Stevia extract from, inter alia, a Stevia plant. Plant-derived components can contribute to off-flavors and include pigments, lipids, proteins, phenolics, saccharides, spathulenol and other sesquiterpenes, labdane diterpenes, monoterpenes, decanoic acid, 8,1 1 , 14-eicosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, tetracosane, octadecanol, stigmasterol, β- sitosterol, a- and β-amyrin, lupeol, β-amryin acetate, pentacyclic triterpenes, centauredin, quercitin, epi-alpha-cadinol, carophyllenes and derivatives, beta-pinene, beta-sitosterol, and gibberellin. In some embodiments, the plant-derived components referred to herein are non- glycoside compounds.
[00107] As used herein, the terms "detectable amount," "detectable concentration," "measurable amount," and "measurable concentration" refer to a level of steviol glycosides measured in AUC, μΜ/ΟΟ600, mg/L, μΜ, or mM. Steviol glycoside production (i.e. , total, supernatant, and/or intracellular steviol glycoside levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high- performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/ spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR).
[00108] As used herein, the term "undetectable concentration" refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound of an "undetectable concentration" is not present in one or more steviol glycosides or glycosides of a steviol precursor.
[00109] After the recombinant microorganism has been grown in culture for the period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside, steviol and/or one or more steviol glycosides can then be recovered from the culture using various techniques known in the art. Steviol glycosides can be isolated using a method described herein. For example, following fermentation, a culture broth can be centrifuged for 30 min at 7000 rpm at 4°C to remove cells, or cells can be removed by filtration. The cell-free lysate can be obtained, for example, by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris. Mechanical disruption of the dried broth materials can also be performed, such as by sonication. The dissolved or suspended broth materials can be filtered using a micron or sub-micron prior to further purification, such as by preparative chromatography. The fermentation media or cell-free lysate can optionally be treated to remove low molecular weight compounds such as salt; and can optionally be dried prior to purification and re-dissolved in a mixture of water and solvent.
[00110] The supernatant or cell-free lysate can be purified as follows: a column can be filled with, for example, HP20 Diaion resin (aromatic type Synthetic Adsorbent; Supeico) or other suitable non-polar adsorbent or reversed-phase chromatography resin, and an aliquot of supernatant or cell-free lysate can be loaded on to the column and washed with water to remove the hydrophilic components. The steviol glycoside product can be eluted by stepwise incremental increases in the solvent concentration in water or a gradient from, e. g. , 0% → 100% methanol). The levels of steviol glycosides, glycosylated enf-kaurenol, and/or glycosylated en/-kaurenoic acid in each fraction, including the flow-through, can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization. For example, steviol glycosides can be isolated by methods not limited to ion exchange chromatography, reversed-phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon columns and/or decoloring steps.
[00111] As used herein, the terms "or" and "and/or" is utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." In some embodiments, "and/or" is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In some embodiments, "and/or" is used to refer to production of steviol glycosides and/or steviol glycoside precursors. In some embodiments, "and/or" is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced. In some embodiments, "and/or" is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced through the following steps: culturing a recombinant microorganism, synthesizing one or more steviol glycosides in a recombinant microorganism, and/or isolating one or more steviol glycosides.
Functional Homologs
[00112] Functional homologs of the polypeptides described above are also suitable for use in producing steviol glycosides in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
[00113] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non- redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol glycoside biosynthesis polypeptides, e.g. , conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
[00114] Conserved regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al. , Nucl. Acids Res., 26:320-322 (1998); Sonnhammer e/ a/. , Proteins, 28:405-420 (1997); and Bateman et al. , Nucl. Acids Res., 27:260- 262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs. [00115] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
[00116] For example, polypeptides suitable for producing steviol in a recombinant host include functional homologs of UGTs.
[00117] Methods to modify the substrate specificity of, for example, a UGT, are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et al., 2009, Phytochemistry 70: 325-347.
[00118] A candidate sequence typically has a length that is from 80% to 250% of the length of the reference sequence, e.g. , 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g. , 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, or 120% of the length of the reference sequence, or any range between. A % sequence identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g. , a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program Clustal Omega (version 1.2.1 , default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res. 31 (13):3497-500.
[00119] ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: % age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1 ; window size: 5; scoring method:% age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; residue-specific gap penalties: on. The ClustalW output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk clustalw).
[00120] To determine a % sequence identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the% sequence identity value can be rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[00121] It will be appreciated that functional UGT proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. In some embodiments, UGT proteins are fusion proteins. The terms "chimera," "fusion polypeptide," "fusion protein," "fusion enzyme," "fusion construct," "chimeric protein," "chimeric polypeptide," "chimeric construct," and "chimeric enzyme" can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins.
[00122] In some embodiments, a chimeric enzyme is constructed by joining the C-terminal of a first polypeptide ProteinA to the N-terminal of a second polypeptide ProteinB through a linker "b," i.e., "ProteinA-b-ProteinB." In some aspects, the linker of a chimeric enzyme may be the amino acid sequence "KLVK." In some aspects, the linker of a chimeric enzyme may be the amino acid sequence "RASSTKLVK." In some aspects, the linker of a chimeric enzyme may be the amino acid sequence "GGGGS." In some aspects, the linker of a chimeric enzyme may be two repeats of the amino acid sequence "GGGGS" (i.e. , "GGGGSGGGGS"). In some aspects, the linker of a chimeric enzyme may be three repeats of the amino acid sequence "GGGGS." In some aspects, the linker of a chimeric enzyme is a direct bond between the C-terminal of a first polypeptide and the N-terminal of a second polypeptide. In some embodiments, a chimeric enzyme is constructed by joining the C-terminal of a first polypeptide ProteinA to the N-terminal of a second polypeptide ProteinB through a linker "b," i.e. , "ProteinA-b-ProteinB" and by joining the C-terminal of the second polypeptide ProteinB to the N-terminal of a third polypeptide ProteinC through a second linker "d," i.e. , "ProteinA-b-ProteinB-d-ProteinC.
[00123] In some embodiments, a nucleic acid sequence encoding a UGT polypeptide (e.g. , a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group) can include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation (e.g. , to facilitate purification or detection), secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non- limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and Flag™ tag (Kodak, New Haven, CT). Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
[00124] In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term "domain swapping" is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein. In some embodiments, a UGT polypeptide (e.g. , a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group) is altered by domain swapping.
[00125] In some embodiments, a fusion protein is a protein altered by circular permutation, which consists in the covalent attachment of the ends of a protein that would be opened elsewhere afterwards. Thus, the order of the sequence is altered without causing changes in the amino acids of the protein. In some embodiments, a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order. The step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
Steviol and Steviol Glycoside Biosynthesis Nucleic Acids
[00126] A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
[00127] In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism. A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g. , non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. "Regulatory region" refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
[00128] The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
[00129] One or more genes can be combined in a recombinant nucleic acid construct in "modules" useful for a discrete aspect of steviol and/or steviol glycoside production. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species. For example, a steviol biosynthesis gene cluster, or a UGT gene cluster, can be combined in a polycistronic module such that, after insertion of a suitable regulatory region, the module can be introduced into a wide variety of species. As another example, a UGT gene cluster can be combined such that each UGT coding sequence is operably linked to a separate regulatory region, to form a UGT module. Such a module can be used in those species for which monocistronic expression is necessary or desirable. In addition to genes useful for steviol or steviol glycoside production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
[00130] It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
[00131] In some cases, it is desirable to inhibit one or more functions of an endogenous polypeptide in order to divert metabolic intermediates towards steviol or steviol glycoside biosynthesis. For example, it may be desirable to downregulate synthesis of sterols in a yeast strain in order to further increase steviol or steviol glycoside production, e.g., by downregulating squalene epoxidase. As another example, it may be desirable to inhibit degradative functions of certain endogenous gene products, e.g., glycohydrolases that remove glucose moieties from secondary metabolites or phosphatases as discussed herein. In such cases, a nucleic acid that overexpresses the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain. Alternatively, mutagenesis can be used to generate mutants in genes for which it is desired to increase or enhance function.
Host Microorganisms
[00132] Recombinant hosts can be used to express polypeptides for producing steviol glycosides, including, but not limited to, a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal cell, an algal cell, an archaeal cell or a bacterial cell.
[00133] A number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g. , gram-negative bacteria, yeast, and fungi. A species and strain selected for use as a steviol glycoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
[00134] Typically, the recombinant microorganism is grown in a fermenter at a temperature(s) for a period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture. Depending on the particular microorganism used in the method, other recombinant genes such as isopentenyl biosynthesis genes and terpene synthase and cyclase genes may also be present and expressed. Levels of substrates and intermediates, e.g., isopentenyl diphosphate, dimethylallyl diphosphate, GGPP, en/-kaurene and en/-kaurenoic acid, can be determined by extracting samples from culture media for analysis according to published methods.
[00135] In some aspects, the recombinant microorganism is grown in a deep well plate. It will be understood that while data on production of steviol glycosides by the recombinant microorganism grown in deep well cultures, in some aspects, may be more easily collected than that in fermentation cultures, the small culture volume of the deep well (e.g. , 1 ml or 0.5 ml) can effect differences in the environment of the microorganism and, therefore its efficiency and effectiveness in producing steviol glycosides. For example, nutrient availability, cellular waste product buildup, pH, temperature, agitation, and aeration may differ significantly between fermentation and deep well cultures. Accordingly, uptake of nutrients or other enzyme substrates may vary, affecting the cellular metabolism (e.g., changing the amount and/or profile of products accumulated by a recombinant microorganism). See, e.g. , Duetz, Trends Microbiol 15(10):469-75 (2007).
[00136] Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the steviol glycosides. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose-comprising polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
[00137] It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate steviol and/or steviol glycosides.
[00138] Alternatively, the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g. , steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, RebA. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00139] Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable. However, it will be appreciated that other species can be suitable to express polypeptides for the producing steviol glycosides.
[00140] For example, suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia (formally known as Hansuela), Scheffersomyces, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces, Humicola, Issatchenkia, Brettanomyces, Yamadazyma, Lachancea, Zygosaccharomyces, Komagataella, Kazachstania, Xanthophyllomyces, Geotrichum, Blakeslea, Dunaliella, Haematococcus, Chlorella, Undaria, Sargassum, Laminaria, Scenedesmus, Pachysolen, Trichosporon, Acremonium, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Pachysolen, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasmsonia, Thermoascus, Thielavia, Tolypocladium, Kloeckera, Pachysolen, Schwanniomyces, Trametes, Trichoderma, Acinetobacter, Nocardia, Xanthobacter, Streptomyces, Erwinia, Klebsiella, Serratia, Pseudomonas, Salmonella, Choroflexus, Chloronema, Chlorobium, Pelodictyon, Chromatium, Rhode-spirillum, Rhodobacter, Rhodomicrobium, or Yarrowia.
[00141] Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastons, Pichia kudriavzevii, Cybehindnera jadinii, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Issatchenkia orientalis, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Hansuela polymorpha, Brettanomyces anomalus, Yamadazyma philogaea, Fusarium fujikuroilGibberella fujikuroi, Candida utilis, Candida glabrata, Candida krusei, Candida revkaufi, Candida pulcherrima, Candida tropicalis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla, Candida albicans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillius licheniformis, Bacillus puntis, Bacillius megaterium, Bacillius halofurans, Baciilius punilus, Serratia marcessans, Pseudomonas aeruginosa, Salmonella typhimurium, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis, Salmonella typhi, Choroflexus aurantiacus, Chloronema gigateum, Chlorobium limicola, Pelodictyon luteolum, Chromatium okenii, Rhode-spirillum rubrum, Rhodobacter spaeroides, Rhodobacter capsulatus, Rhodomicrobium vanellii, Pachysolen tannophilus, Trichosporon beigelii, and Yarrowia lipolytica.
[00142] In some embodiments, a microorganism can be a prokaryote such as Escherichia bacteria cells, for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Comebacterium bacteria cells; Acetobacter bacteria cells; Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
[00143] In some embodiments, a microorganism can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp. , Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.
[00144] In some embodiments, a microorganism can be a fungi from the genera including but not limited to Acremonium, Arxula, Agaricus, Aspergillus, Agaricus, Aureobasidium, Brettanomyces, Candida, Cryptococcus, Corynascus, Chrysosporium, Debaromyces, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus, Schizophyllum, Schizosaccharomyces, Sordaria, Scheffersomyces, Talaromyces, Rhodotorula, Rhodosporidium, Rasmsonia, Zygosaccharomyces, Thermoascus, Thielavia, Trichosporon, Tolypocladium, Trametes, and Trichoderma. Fungal species include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla.
[00145] In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Geotrichum Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, Yamadazyma philogaea, Lachancea kluyveri, Kodamaea ohmeri, or S. cerevisiae.
Agaricus, Gibberella, and Phanerochaete spp.
[00146] Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture. Thus, the terpene precursors for producing large amounts of steviol glycosides are already produced by endogenous genes. Thus, modules comprising recombinant genes for steviol glycoside biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
Arxula adeninivorans (Blastobotrys adeninivorans)
[00147] Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42°C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
Rhodotorula sp.
[00148] Rhodotorula is unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al, 201 1 , Process Biochemistry 46(1):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41 :312-7).
Schizosaccharomyces spp.
[00149] Schizosaccharomyces is a genus of fission yeasts. Similar to S. cerevisiae, Schizosaccharomyces is a model organism in the study of eukaryotic cell biology. It provides an evolutionary distant comparison to S. cerevisiae. Species include but are not limited to S. cryophilius and S. pombe. (See Hoffman et al., 2015, Genetics. 201 (2):403-23).
Humicola spp.
[00150] Humicola is a genus of filamentous fungi. Species include but are not limited to H. alopallonella and H. siamensis.
Brettanomyces spp. [00151] Bretianomyces is a non-spore forming genus of yeast. It is from the Saccharomycetaceae family and commonly used in the brewing and wine industries. Bretianomyces produces several sensory compounds that contribute to the complexity of wine, specifically red wine. Bretianomyces species include but are not limited to B. bruxellensis and B. claussenii. See, e.g., Fugelsang et al. , 1997, Wine Microbiology.
Trichosporon spp.
[00152] Trichosporon is a genus of the fungi family. Trichosporon species are yeast commonly isolated from the soil, but can also be found in the skin microbiota of humans and animals. Species include, for example but are not limited to, T. aquatile, T. beigelii, and T. dermatis.
Debaromyces spp.
[00153] Debaromyces is a genus of the ascomycetous yeast family, in which species are characterized as a salt-tolerant marine species. Species include but are not limited to D. hansenii and D. hansenius.
Physcomitrella spp.
[00154] Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
Saccharomyces spp.
[00155] Saccharomyces is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms. Examples of Saccharomyces species include S. castellii, also known as Naumovozyma castelli.
Zygosaccharomyces spp.
[00156] Zygosaccharomyces is a genus of yeast. Originally classified under the Saccharomyces genus it has since been reclassified. It is widely known in the food industry because several species are extremely resistant to commericially used food preservation techniques. Species include but are not limited to Z bisporus and Z cidn. (See Bamett et al, Yeasts: Characteristics and Identification, 1983).
Geotrichum spp.
[00157] Geotrichum is a fungi commonly found in soil, water and sewage worldwide. It's often identified in plants, cereal and diary products. Species include, for example but are not limited to, G. candidum and G. klebahnii (see Carmichael et al. , Mycologica, 1957, 49(6):820-830).
Kazachstania sp
[00158] Kazachstania is a yeast genus in the family Sacchromycetaceae.
Torulaspora spp.
[00159] Torulaspora is a genus of yeasts and species include but are not limited to T. franciscae and T. globosa.
Aspergillus spp.
[00160] Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing steviol glycosides.
Yarrowia lipolytica
[00161] Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g., alkanes, fatty acids, and oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorgamism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g., Nicaud, 2012, Yeas/ 29(10):409-18; Beopoulos et al., 2009, Biochimie 91 (6):692-6; Bankar et al. , 2009, Appl Microbiol Biotechnol. 84(5):847- 65.
Rhodosporidium toruloides
[00162] Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid- production pathways (See e.g. Zhu et al., 2013, Nature Commun. 3:1 1 12; Ageitos et al. , 201 1 , Applied Microbiology and Biotechnology 90(4) : 1219-27) .
Candida boidinii
[00163] Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g. , Mattanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
Hansenula polymorpha (Pichia angusta)
[00164] Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also, Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu et al., 2014, Virol Sin. 29(6):403-9.
Candida krusei (Issatchenkia orientalis)
[00165] Candida krusei , scientific name Issatchenkia orientalis, is widely used in chocolate production. C. krusei is used to remove the bitter taste of and break down cacao beans. In addition to this species involvement in chocolate production, C. krusei is commonly found in the immunocompromised as a fungal nosocomial pathogen (see Mastromarino et al. , New Microbiolgica, 36:229-238; 2013)
Kluyveromyces lactis
[00166] Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen et al. , 2006, FEMS Yeast Res. 6(3):381-92.
Pichia pastoris
[00167] Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It is also commonly referred to as Komagataella pastoris. It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen et al., 2014, N Biotechnol. 31 (6):532-7.
Scheffersomyces stipitis
[00168] Scheffersomyces stipitis also known as Pichia stipitis is a homothallic yeast found in haploid form. Commonly used instead of S. cerevisiae due to its enhanced respiratory capacity that results from and alternative respiratory system. (See Papini et al, Microbial Cell Factories, 1 1 :136 (2012)).
[00169] In some embodiments, a microorganism can be an insect cell such as Drosophilia, specifically, Drosophilia melanogaster.
[00170] In some embodiments, a microorganism can be an algal cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp.,
[00171] In some embodiments, a microorganism can be a cyanobacterial cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis.
[00172] In some embodiments, a microorganism can be a bacterial cell. Examples of bacteria include, but are not limited to, the genenera Bacillus (e.g. , B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus), Acinetobacter, Nocardia, Xanthobacter, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g. , S. marcessans), Pseudomonas (e.g. , P. aeruginosa), Salmonella (e.g., S. typhimurium, and S. typhi). Bacterial cells may also include, but are not limited to, photosynthetic bacteria (e.g. , green non-sulfur bacteria (e.g. , Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g. , C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g. , P. luteolum), purple sulfur bacteria (e.g. , Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii)).
E. coli
[00173] E. coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
[00174] It can be appreciated that the recombinant host cell disclosed herein can comprise a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal eel from Aspergillus genus; a yeast cell from Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, and S. carlsbergensis), Schizosaccharomyces (e.g. , S. pombe), Yarrowia (e.g., Y. lipolytica), Candida (e.g., C. glabrata, C. albicans, C. krusei, C. revkaufi, C. pulcherrima, Candida tropicalis, C. utilis, and C. boidinii), Ashbya (e.g. , A. gossypii), Cyberlindnera (e.g., C. jadinii), Pichia (e.g. , P. pastoris and P. kudriavzevii), Kluyveromyces (e.g., K. lactis), Hansenual (e.g., H. polymorpha), Arxula (e.g., A. adeninivorans), Xanthophyllomyces (e.g., X. dendrorhous), Issatchenkia (e.g. , I. orientali), Torulaspora (e.g. , T. franciscae and T. globosa), Geotrichum (e.g., G. candidum and G. klebahni), Zygosaccharomyces (e.g., Z. bisporus and Z. cidri), Yamadazyma (e.g., Y. philogaea), Lanchancea (e.g., L. kluyveri), Kodamaea (e.g., K. ohmeri), Brettanomyces (e.g., B. anomalus), Trichosporon (e.g. , T. aquatile, T. beigelii, and T. dermatis), Debaromyces (e.g. , D. hansenuis and D. hansenii), Scheffersomyces (e.g., S. stipis), Rhodosporidium (e.g., R. toruloides), Pachysolen (e.g., P. tannophilus), and Physcomitrella, Rhodotorula, Kazachstania, Gibberella, Agaricus, and Phanerochaete genera; an insect cell including, but not limited to, Drosophilia melanogaster, an algal cell including, but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis species; or a bacterial cell from Bacillus genus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, and B. pumilus) Acinetobacter, Nocardia, Xanthobacter genera, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g. , P. aeruginosa), Salmonella (e.g., S. typhimurium and S. typhi), and further including, Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum)), purple sulfur bacteria (e.g., Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g. , Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g. , R. sphaeroides and R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii).
Steviol Glycoside Compositions
[00175] Steviol glycosides do not necessarily have equivalent performance in different food systems. It is therefore desirable to have the ability to direct the synthesis to steviol glycoside compositions of choice. Recombinant hosts described herein can produce compositions that are selectively enriched for specific steviol glycosides (e.g., RebD or RebM) and have a consistent taste profile. As used herein, the term "enriched" is used to describe a steviol glycoside composition with an increased proportion of a particular steviol glycoside, compared to a steviol glycoside composition (extract) from a stevia plant. Thus, the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the sweetening profile desired for a given food product and that have a proportion of each steviol glycoside that is consistent from batch to batch. In some embodiments, hosts described herein do not produce or produce a reduced amount of undesired plant by-products found in Stevia extracts. Thus, steviol glycoside compositions produced by the recombinant hosts described herein are distinguishable from compositions derived from Stevia plants.
[00176] The amount of an individual steviol glycoside (e.g. , RebA, RebB, RebD, or RebM) accumulated can be from about 1 to about 7,000 mg/L, e.g., about 1 to about 10 mg/L, about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50 mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100 to about 1 ,500 mg/L, or about 200 to about 1 ,000 mg/L, at least about 1 ,000 mg/L, at least about 1 ,200 mg/L, at least about at least 1 ,400 mg/L, at least about 1 ,600 mg/L, at least about 1 ,800 mg/L, at least about 2,800 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of an individual steviol glycoside can exceed 7,000 mg/L. The amount of a combination of steviol glycosides (e.g. , RebA, RebB, RebD, or RebM) accumulated can be from about 1 mg/L to about 7,000 mg/L, e.g., about 200 to about 1 ,500, at least about 2,000 mg/L, at least about 3,000 mg/L, at least about 4,000 mg/L, at least about 5,000 mg/L, at least about 6,000 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of a combination of steviol glycosides can exceed 7,000 mg/L. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.
[00177] It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant microorganisms rather than a single microorganism. When a plurality of recombinant microorganisms is used, they can be grown in a mixed culture to produce steviol and/or steviol glycosides. For example, a first microorganism can comprise one or more biosynthesis genes for producing a steviol glycoside precursor, while a second microorganism comprises steviol glycoside biosynthesis genes. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00178] Alternatively, the two or more microorganisms each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as RebA. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00179] Steviol glycosides and compositions obtained by the methods disclosed herein can be used to make food products, dietary supplements and sweetener compositions. See, e.g., WO 201 1/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
[00180] For example, substantially pure steviol or steviol glycoside such as RebM or RebD can be included in food products such as ice cream, carbonated 2s, fruit juices, yogurts, baked goods, chewing gums, hard and soft candies, and sauces. Substantially pure steviol or steviol glycoside can also be included in non-food products such as pharmaceutical products, medicinal products, dietary supplements and nutritional supplements. Substantially pure steviol or steviol glycosides may also be included in animal feed products for both the agriculture industry and the companion animal industry. Alternatively, a mixture of steviol and/or steviol glycosides can be made by culturing recombinant microorganisms separately, each producing a specific steviol or steviol glycoside, recovering the steviol or steviol glycoside in substantially pure form from each microorganism and then combining the compounds to obtain a mixture comprising each compound in the desired proportion. The recombinant microorganisms described herein permit more precise and consistent mixtures to be obtained compared to current Stevia products.
[00181] In another alternative, a substantially pure steviol or steviol glycoside can be incorporated into a food product along with other sweeteners, e.g., saccharin, dextrose, sucrose, fructose, erythritol, aspartame, sucralose, monatin, or acesulfame potassium. The weight ratio of steviol or steviol glycoside relative to other sweeteners can be varied as desired to achieve a satisfactory taste in the final food product. See, e.g. , U.S. 2007/012831 1 . In some embodiments, the steviol or steviol glycoside may be provided with a flavor (e.g. , citrus) as a flavor modulator.
[00182] Compositions produced by a recombinant microorganism described herein can be incorporated into food products. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a food product in an amount ranging from about 20 mg steviol glycoside/kg food product to about 1800 mg steviol glycoside/kg food product on a dry weight basis, depending on the type of steviol glycoside and food product. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a dessert, cold confectionary (e.g. , ice cream), dairy product (e.g., yogurt), or beverage (e.g., a carbonated beverage) such that the food product has a maximum of 500 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a baked good (e.g. , a biscuit) such that the food product has a maximum of 300 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a sauce (e.g. , chocolate syrup) or vegetable product (e.g., pickles) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into bread such that the food product has a maximum of 160 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a hard or soft candy such that the food product has a maximum of 1600 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a processed fruit product (e.g. , fruit juices, fruit filling, jams, and jellies) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. In some embodiments, a steviol glycoside composition produced herein is a component of a pharmaceutical composition. See, e.g. , Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org.; EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), "Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive," 2010, EFSA Journal 8(4): 1537; U.S. Food and Drug Administration GRAS Notice 323; U.S Food and Drug Administration GRAS Notice 329; WO 2011/037959; WO 2010/146463; WO 201 1/046423; and WO 201 1/056834.
[00183] For example, such a steviol glycoside composition can have from 90-99 weight % RebA and an undetectable amount of stevia plant-derived contaminants, and be incorporated into a food product at from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
[00184] Such a steviol glycoside composition can be a RebB-enriched composition having greater than 3 weight % RebB and be incorporated into the food product such that the amount of RebB in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebB-enriched composition has an undetectable amount of stevia plant-derived contaminants.
[00185] Such a steviol glycoside composition can be a RebD-enriched composition having greater than 3 weight % RebD and be incorporated into the food product such that the amount of RebD in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebD-enriched composition has an undetectable amount of stevia plant-derived contaminants.
[00186] Such a steviol glycoside composition can be a RebE-enriched composition having greater than 3 weight % RebE and be incorporated into the food product such that the amount of RebE in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebE-enriched composition has an undetectable amount of stevia plant-derived contaminants.
[00187] Such a steviol glycoside composition can be a RebM-enriched composition having greater than 3 weight % RebM and be incorporated into the food product such that the amount of RebM in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebM-enriched composition has an undetectable amount of stevia plant-derived contaminants. [00188] In some embodiments, a substantially pure steviol or steviol glycoside is incorporated into a tabletop sweetener or "cup-for-cup" product. Such products typically are diluted to the appropriate sweetness level with one or more bulking agents, e.g., maltodextrins, known to those skilled in the art. Steviol glycoside compositions enriched for RebA, RebB, RebD, RebE, or RebM, can be package in a sachet, for example, at from 10,000 to 30,000 mg steviol glycoside/kg product on a dry weight basis, for tabletop use. In some embodiments, a steviol glycoside produced in vitro, in vivo, or by whole cell bioconversion
[00189] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
[00190] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Example 1. UGT74G1 Variant Expression
[00191] A set of 154 possible mutations at 149 positions of the UGT74G1 polypeptide of SEQ ID NO:4 was identified through modeling as described above in "Functional Homologs" (See Table 1). A library of 179 UGT74G1 variants (i.e., functional homologs) including one or more identified mutations was prepared (SEQ ID NO:3, SEQ ID NO:4). See Table 2.
Table 1. UGT74G1 Mutation Set
N51K E176D V285A L385F
Q67E L179S H287L D387E
A68T I180F V292M E388D
C73F L181V I295L I389V
E75D N183D I295M L390W
M79A H184P T296A G391K
E83D E185G R297W V396A
E83K Q186S A298G N401K
S84A I187Y I300K G407E
L86I Q188P D301N N408E
E87D S189A D303N L409I
T88R W191F 1310V A410E
K90W S192D K311R S411D
Q91E Q193M K313S M415E
S96T M194V E315Q 1416V
D99E L195V G316A E419G
E107S A200S E320K I424E
T110P Q204K L322F R426K
1111V F209L V325E K427E
A113C N211H I326T D434E
1115V S212T T328S N448K
M119F K215E G329E D449N
T120L 1221V L332I S455A
E121P E222D I333V E456K
V123A T224M A335S I458V
I128K V232T K338P
E129Q L237I D341E
G135A D247E E346P
S136A N252Y E346K
N255S S347A
Table 2. UGT74G1 Variant Library
3 A200S E129Q Q140N
4 L390W N266K S192D
5 E320K N266K M194V
6 M265I I364L I187Y
7 E315Q E129Q S192D
8 E222D N408E Q140N
9 M1 19F N255S A298G
10 D269N R426K S146N
11 E185G A298G Q193M
12 V232T E388D Q140N
13 G316A V325E I187Y
14 L409I D301 N H184P
15 S189A S146N I389V
16 V143A 1416V H184P
17 S455A Q193M E346K
18 R297W S347A E346K
19 D341 E Q193M L332I
20 D434E T380S I389V
21 T328S A280S L332I
22 Q375L E419G R426K
23 1221V K338P I295M
24 T296A I128K L332I
25 D449N G407E H287L
26 T1 10P K338P E346P
27 E121 P 1416V H287L
28 L284M I424E E346K
29 K427E K338P I424E
30 D303N N408E I300K
31 D247E D301 N E346P
32 F376W N448K I128K
33 K313S I187Y I128K
34 G329E G407E N266E
35 11 1 1V M415E H287L 36 G391K T380S I389V
37 V396A N448K S347A
38 1310V V371I N266E
39 Y257F N401K W191F
40 S136A I364L I424E
41 V292M E419G I364L
42 D99E L322F S192D
43 C73F S146N T380S
44 A259P V371I K90W
45 T49I A280S A113C
46 V123A M194V T88R
47 L181V A280S L86I
48 N252Y E129Q F30L
49 A68T L322F A113C
50 S212T F357W F30L
51 E75D I300K F357W
52 A335S G407E Q91E
53 I16L A113C M415E
54 G31S N255S I295M
55 S377T E388D L86I
56 I180F H184P E83K
57 Q188P N408E E83K
58 K311R W191F F27M
59 L195V L20A E346P
60 M79A V325E M415E
61 Q67E N401K D301N
62 S84A S347A I295M
63 A141S F27M V371I
64 L179S N266K E83K
65 Q186S W191F T88R
66 A410E A298G L20A
67 K311R W191F F27M
68 V285A E388D L20A 69 E176D L322F K90W
70 F169L K90W N266E
71 F18Y 1416V F27M
72 E87D Q91 E I300K
73 E274G L86I F30L
74 E83D R426K Q91 E
75 S96T V325E T88R
76 E176D F357W
77 S377T
78 M1 19F
79 E456K
80 L181V
81 L385F
82 N183D
83 E176D
84 F209L
85 N21 1 H
86 V143A
87 R297W
88 A410E
89 L390W
90 N252Y
91 S212T
92 V232T
93 11 15V
94 G329E
95 T224M
96 I295L
97 T328S
98 L409I
99 D387E
100 D449N 101 V123A
102 M373V
103 V285A
104 Q204K
105 S189A
106 D247E
107 G135A
108 11 1 1V
109 T120L
110 G316A
111 Q173E
112 V166L
113 1221V
114 L147I
115 F376W
116 L284M
117 E162T
118 Q375L
119 S136A
120 E315Q
121 I333V
122 M265I
123 A141 S
124 E107S
125 E185G
126 V396A
127 L237I
128 Q186S
129 E320K
130 A200S
131 L195V 132 Q188P
133 Y257F
134 D269N
135 D341E
136 D434E
137 K313S
138 L179S
139 S455A
140 E263A
141 K311R
142 A259P
143 T110P
144 V292M
145 I326T
146 T296A
147 E222D
148 G391K
149 K215E
150 1310V
151 I156L
152 D303N
153 E121P
154 V370M
155 K427E
156 I180F
157 E274G
158 I458V
159 A335S
160 S411D
161 F169L
162 L15V 163 F18Y
164 M79A
165 E87D
166 G31 S
167 E83D
168 N51 K
169 E75D
170 T49I
171 D99E
172 S96T
173 C73F
174 S84A
175 A68T
176 Q67E
177 I16L
178 I28L
179 G31A
[00192] Competent E. coli cells were transformed with vectors expressing the UGT74G1 variants of Table 2. After transformation, 80 μΙ_ of each culture was transferred into 800 μΙ_ of standard lysogeny broth (LB) medium containing 100 μg mL ampicillin and 50 μg mL chloramphenicol and incubated at 37°C for 18 hours, shaking at 300 rpm. Glycerol stocks of the transformed cells (25% glycerol) were prepared and stored at -80°C.
[00193] 1 mL of auto-induction pre-culture medium containing 100 μg mL ampicillin and 50 μg mL chloramphenicol was inoculated with 10 μί of a glycerol stock prepared as described in the previous paragraph and incubated in a 96-well plate for 20 hours at 25°C, shaking at 300 rpm. 1 mL of auto-induction medium containing 100 μg mL ampicillin and 50 μg mL chloramphenicol was then inoculated with an amount of pre-culture sample sufficient to provide an initial OD of 0.2 (~20-25 μί of pre-culture) and incubated for 18 hours at 25°C, shaking at 300 rpm. Cells were then pelleted by centrifugation at 3500 rpm for 15 minutes at 4°C. The supernatant was discarded by inverting the plate and subsequently tapping the inverted plate on tissue paper. Pellets were then frozen at -80°C for at least 15 minutes. [00194] After thawing cell pellets in a water bath at room temperature, binding and lysozyme buffer with protease inhibitor (Tris-HCI 20 mM pH8, NaCI 0.5 M, Imidazole 20 mM, Lysozyme 0.2 mg/ml, DNase I 20 μg ml, MgCI2 1 mM, protease inhibitor complete mini-tablet 1x) was added to the wells in an amount of 1 mL per 100 mg cells (~250 μΙ_). Cells were resuspended on an orbital shaker at 300 rpm for 15 minutes at 20°C, and then incubated for 2 hours at 4°C. After incubation, cells were lysed via one or more freeze-thaw cycles, and then clarified by centrifugation at 3000 g for 15 minutes at 4°C. The supernatant was transferred to a clean 96- well plate and diluted with glycerol (40% glycerol). Samples were stored at -20°C.
Example 2. UGT74G1 Variant Activity Assay
[00195] 60 μΙ_ of reaction mixtures in 96-well plates prepared according to Table 3 were incubated at 30°C, shaking at 75-100 rpm for 2h. The reaction was then quenched by diluting the mixture 1 :5 in pure methanol and centrifuged at 3500 rpm for 15 minutes. The supernatant was isolated and stored at -80°C until LC-MS analysis.
Table 3. Activity Assay Reaction Mixture
[00196] A 5 μΙ_ sample of the quenched reaction mixture was injected into a Water Acquity UPLC system (Milford, USA) coupled to a Bruker mictoTOF-Q II mass detector (Bremen, Germany). Separation of the compounds was achieved on a Waters Acquity UPLC® BEH C18 column (1.7 μηη, 2.1 mm x 50 mm) kept at 55°C, using a gradient of two mobile phases: A (water with 5 mM ammonium formate, pH 9.0) and B (acetonitrile) at a flow of 0.6 mL/min. The gradient profile consisted of 25% B for 0.3 minutes, a linear gradient from 25% B to 85% B over 2 minutes, a 100% B wash for 1 minute, and finally 35% B for 0.6 minutes. The mass analyzer was equipped with electrospray ionization (ESI) source and operated in negative mode. The capillary voltage was 3.5kV, the source was kept at 180°C, and the desolvation gas flow and nebulizer pressure were 8 L/min and 1.6 bar, respectively.
[00197] Compounds of interest were tracked in MS full scan mode (120-800 m/z range) and quantification was performed post-acquisition by extracting ions from the total ion chromatogram. Extracted-ion chromatograms (EICs) provided semi-quantification of steviol-13- O-glucoside (13-SMG) (525.3 m/z [M+Fa-H] ), kaurenoic acid (KA) (301.2 m/z [M-H]"), rubusoside (rubu) (687.3 m/z [M+Fa-H]"), and steviol (317.2 m/z [M-H]"), using one-point calibration with 10 μΜ authentic standards using Bruker QuantAnalysis software. Steviol-19-O- glucoside (19-SMG) (525.3 [M+Fa-H]") and kaurenoate-19-O-glucoside (19-KMG) (509.3 m/z [M+Fa-H]") concentrations were estimated as 13-SMG equivalents, using corrected response factors. Results are shown in Table 4.
Table 4. UGT74G1 Variant Activity
Variant Concentration (μΜ)
No. 13-SMG Steviol KA Rubu 19-SMG* 19-KMG*
39 2.53 2.52 12.91 22.32 23.86 79.53
42 3.38 19.73 82.66 28.77 1 1 .20 18.37
43 3.24 18.61 82.21 27.82 9.52 17.38
44 2.89 14.60 71.56 24.54 12.04 21 .30
45 3.06 1 1.95 61.62 23.70 15.42 29.74
46 3.18 17.69 72.26 22.07 7.72 15.17
50 2.34 6.37 44.47 22.18 18.63 43.54
52 5.95 6.00 22.43 32.80 32.29 1 10.42
55 13.51 18.39 73.61 10.63 5.78 9.68
56 3.04 4.34 26.78 23.23 22.25 67.34
60 4.24 4.61 18.73 21 .84 21 .98 77.35
61 3.44 3.82 15.99 22.99 22.50 77.44
62 3.12 3.68 17.19 21 .93 22.94 77.94
63 3.37 3.08 15.05 23.00 23.67 79.82
64 3.06 3.34 16.67 21 .64 23.09 78.07
65 2.81 3.43 13.66 23.56 23.68 78.43
66 2.68 2.91 14.22 22.88 22.18 81 .72
67 2.92 2.61 13.09 23.06 23.23 82.22
68 3.20 3.13 13.73 22.02 22.86 78.44
69 28.66 28.17 99.32 0.00 0.00 0.00
70 27.93 25.44 99.83 0.00 0.00 0.00
71 4.46 5.17 26.51 24.49 24.73 75.00
72 5.38 5.13 22.50 25.62 25.30 82.77
73 3.05 3.73 26.10 22.31 21 .39 63.87
74 3.57 3.32 15.94 27.46 25.24 90.20
75 4.15 3.93 19.42 22.87 22.67 74.63
76 4.02 4.59 26.21 21 .32 22.61 64.74
77 9.43 21.10 89.01 21 .77 7.56 9.77
78 7.50 22.54 88.99 20.52 3.98 6.03
79 7.72 22.20 89.16 18.67 3.73 5.41
80 2.79 7.03 51.82 25.74 20.71 47.77 Variant Concentration (μΜ)
No. 13-SMG Steviol KA Rubu 19-SMG* 19-KMG*
81 2.40 6.90 50.09 23.24 18.63 41 .50
82 7.18 6.97 27.31 34.42 17.24 53.56
83 16.67 23.33 86.84 1 1 .50 1.54 2.24
84 4.09 4.23 27.50 22.93 23.07 64.68
87 5.04 4.95 22.57 24.34 22.85 71 .84
88 4.71 5.05 19.64 26.62 26.73 91 .63
89 5.02 5.06 19.78 26.61 25.35 88.49
90 4.01 3.94 22.04 24.12 22.94 74.25
94 4.55 5.01 20.99 24.69 24.73 83.53
95 3.96 4.28 17.84 26.80 26.00 88.13
98 3.36 4.07 17.02 27.03 25.99 88.04
99 3.59 3.80 16.1 1 27.60 25.94 89.85
100 3.98 4.26 21.80 22.37 23.52 72.60
103 4.01 4.34 17.46 25.87 24.72 83.42
104 3.72 4.08 16.68 26.42 27.23 91 .74
107 4.22 4.65 20.34 23.72 22.30 75.33
108 4.05 4.21 19.38 24.24 23.42 79.05
110 3.05 3.42 15.34 26.99 27.24 92.27
111 4.62 4.91 19.76 23.59 23.89 81 .07
113 2.82 3.65 18.67 24.09 23.54 79.07
114 4.14 4.22 18.60 24.32 24.90 85.59
115 5.16 5.09 18.96 23.57 23.45 77.95
116 3.02 3.65 17.47 24.52 24.33 81 .05
117 4.27 4.26 18.71 23.66 24.46 82.38
118 3.36 3.28 16.53 24.74 23.83 77.63
121 4.46 4.73 18.35 24.31 23.66 84.87
122 3.04 3.96 16.01 25.28 25.16 86.71
123 3.69 3.63 17.05 24.57 23.66 83.40
124 3.55 4.26 19.29 22.12 22.58 73.14
129 3.04 3.15 15.76 25.55 23.29 84.24 Variant Concentration (μΜ)
No. 13-SMG Steviol KA Rubu 19-SMG* 19-KMG*
130 3.78 3.88 15.91 24.18 25.77 86.22
131 3.63 4.00 16.07 24.1 1 24.95 84.21
132 3.18 3.42 16.69 23.65 23.83 80.85
137 2.51 2.85 14.32 25.23 24.84 85.27
138 3.40 3.36 16.04 23.90 23.14 80.14
139 2.78 3.10 14.81 25.22 24.64 87.95
143 3.05 3.45 15.39 24.22 23.43 84.29
144 4.01 4.42 16.59 22.99 23.26 81 .73
145 3.17 3.58 15.45 23.43 25.04 86.51
146 3.18 3.53 14.27 23.86 24.50 82.34
150 2.99 3.81 15.75 22.67 23.64 81 .30
151 3.03 3.85 16.03 22.54 22.73 80.26
152 3.44 3.65 15.70 22.86 22.64 80.63
153 2.83 2.72 13.74 24.30 23.98 86.19
154 2.97 3.38 15.64 22.23 22.64 78.53
155 3.02 3.75 15.35 22.32 23.06 80.09
156 2.89 3.03 14.86 22.44 22.67 77.55
161 26.48 25.52 95.94 1.21 0.21 0.18
162 1.96 14.27 69.61 23.43 10.27 16.84
164 4.01 4.05 17.63 26.21 25.30 84.15
166 3.03 3.43 18.76 23.88 23.38 79.46
167 2.87 3.35 15.12 25.15 24.54 81 .59
169 3.30 3.58 15.76 25.05 24.19 86.24
170 3.69 3.62 16.20 23.60 23.22 76.74
173 4.48 4.43 18.75 21 .1 1 21 .51 72.60
174 2.71 2.89 14.68 23.55 24.16 80.31
176 3.70 3.68 16.07 22.98 24.34 87.92
177 3.06 3.01 14.09 23.47 22.76 80.44
178 3.25 3.31 15.04 21 .65 24.07 80.23
179 2.75 3.04 14.06 21 .89 22.67 76.74 *estimated as 13-SMG equivalents.
[00198] The results, provided in Table 4, show that UGT74G1 variants produce one or more steviol glycosides or glycosides of a steviol precursor in relative amounts different than those of a wild-type UGT74G1 polypeptide. For example, several of the variants of Table 4 produce rubusoside and 19-SMG in relative amounts different than those of a wild-type UGT74G1 polypeptide (see e.g. , Example 21 of WO 201 1/153378).
[00199] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
Table 5. Sequences disclosed herein.
SEQ ID NO:3
S. rebaudiana
atggcagagc aacaaaagat caaaaagtca cctcacgtct tacttattcc atttcctctg 60 caaggacata tcaacccatt catacaattt gggaaaagat tgattagtaa gggtgtaaag 120 acaacactgg taaccactat ccacactttg aattctactc tgaaccactc aaatactact 180 actacaagta tagaaattca agctatatca gacggatgcg atgagggtgg ctttatgtct 240 gccggtgaat cttacttgga aacattcaag caagtgggat ccaagtctct ggccgatcta 300 atcaaaaagt tacagagtga aggcaccaca attgacgcca taatctacga ttctatgaca 360 gagtgggttt tagacgttgc tatcgaattt ggtattgatg gaggttcctt tttcacacaa 420 gcatgtgttg tgaattctct atactaccat gtgcataaag ggttaatctc tttaccattg 480 ggtgaaactg tttcagttcc aggttttcca gtgttacaac gttgggaaac cccattgatc 540 ttacaaaatc atgaacaaat acaatcacct tggtcccaga tgttgtttgg tcaattcgct 600 aacatcgatc aagcaagatg ggtctttact aattcattct ataagttaga ggaagaggta 660 attgaatgga ctaggaagat ctggaatttg aaagtcattg gtccaacatt gccatcaatg 720 tatttggaca aaagacttga tgatgataaa gataatggtt tcaatttgta caaggctaat 780 catcacgaat gtatgaattg gctggatgac aaaccaaagg aatcagttgt atatgttgct 840 ttcggctctc ttgttaaaca tggtccagaa caagttgagg agattacaag agcacttata 900 gactctgacg taaacttttt gtgggtcatt aagcacaaag aggaggggaa actgccagaa 960 aacctttctg aagtgataaa gaccggaaaa ggtctaatcg ttgcttggtg taaacaattg 1020 gatgttttag ctcatgaatc tgtaggctgt tttgtaacac attgcggatt caactctaca 1080 ctagaagcca tttccttagg cgtacctgtc gttgcaatgc ctcagttctc cgatcagaca 1140 accaacgcta aacttttgga cgaaatacta ggggtgggtg tcagagttaa agcagacgag 1200 aatggtatcg tcagaagagg gaacctagct tcatgtatca aaatgatcat ggaagaggaa 1260 agaggagtta tcataaggaa aaacgcagtt aagtggaagg atcttgcaaa ggttgccgtc 1320 catgaaggcg gctcttcaga taatgatatt gttgaatttg tgtccgaact aatcaaagcc 1380 taa 1383
SEQ ID NO:4 S. rebaudiana
MAEQQKIKKS PHVLLIPFPL QGHINPFIQF GKRLISKGVK TTLVTTIHTL NSTLNHSNTT 60
TTSIEIQAIS DGCDEGGFMS AGESYLETFK QVGSKSLADL IKKLQSEGTT IDAI IYDSMT 120
EWVLDVAIEF GIDGGSFFTQ ACWNSLYYH VHKGLISLPL GETVSVPGFP VLQRWETPLI 180
LQNHEQIQSP WSQMLFGQFA NIDQARWVFT NSFYKLEEEV IEWTRKIWNL KVIGPTLPSM 240
YLDKRLDDDK DNGFNLYKAN HHECMNWLDD KPKESWYVA FGSLVKHGPE QVEEITRALI 300
DSDVNFLWVI KHKEEGKLPE NLSEVIKTGK GLIVAWCKQL DVLAHESVGC FVTHCGFNST 360
LEAISLGVPV VAMPQFSDQT TNAKLLDEIL GVGVRVKADE NGIVRRGNLA SCIKMIMEEE 420
RGVIIRKNAV KWKDLAKVAV HEGGSSDNDI VEFVSELIKA 460
SEQ ID NO:5
S. rebaudiana
atggatgcaa tggctacaac tgagaagaaa ccacacgtca tcttcatacc atttccagca 60 caaagccaca ttaaagccat gctcaaacta gcacaacttc tccaccacaa aggactccag 120 ataaccttcg tcaacaccga cttcatccac aaccagtttc ttgaatcatc gggcccacat 180 tgtctagacg gtgcaccggg tttccggttc gaaaccattc cggatggtgt ttctcacagt 240 ccggaagcga gcatcccaat cagagaatca ctcttgagat ccattgaaac caacttcttg 300 gatcgtttca ttgatcttgt aaccaaactt ccggatcctc cgacttgtat tatctcagat 360 gggttcttgt cggttttcac aattgacgct gcaaaaaagc ttggaattcc ggtcatgatg 420 tattggacac ttgctgcctg tgggttcatg ggtttttacc atattcattc tctcattgag 480 aaaggatttg caccacttaa agatgcaagt tacttgacaa atgggtattt ggacaccgtc 540 attgattggg ttccgggaat ggaaggcatc cgtctcaagg atttcccgct ggactggagc 600 actgacctca atgacaaagt tttgatgttc actacggaag ctcctcaaag gtcacacaag 660 gtttcacatc atattttcca cacgttcgat gagttggagc ctagtattat aaaaactttg 720 tcattgaggt ataatcacat ttacaccatc ggcccactgc aattacttct tgatcaaata 780 cccgaagaga aaaagcaaac tggaattacg agtctccatg gatacagttt agtaaaagaa 840 gaaccagagt gtttccagtg gcttcagtct aaagaaccaa attccgtcgt ttatgtaaat 900 tttggaagta ctacagtaat gtctttagaa gacatgacgg aatttggttg gggacttgct 960 aatagcaacc attatttcct ttggatcatc cgatcaaact tggtgatagg ggaaaatgca 1020 gttttgcccc ctgaacttga ggaacatata aagaaaagag gctttattgc tagctggtgt 1080 tcacaagaaa aggtcttgaa gcacccttcg gttggagggt tcttgactca ttgtgggtgg 1140 ggatcgacca tcgagagctt gtctgctggg gtgccaatga tatgctggcc ttattcgtgg 1200 gaccagctga ccaactgtag gtatatatgc aaagaatggg aggttgggct cgagatggga 1260 accaaagtga aacgagatga agtcaagagg cttgtacaag agttgatggg agaaggaggt 1320 cacaaaatga ggaacaaggc taaagattgg aaagaaaagg ctcgcattgc aatagctcct 1380 aacggttcat cttctttgaa catagacaaa atggtcaagg aaatcaccgt gctagcaaga 1440 aactagttac aaagttgttt cacattgtgc tttctattta agatgtaact ttgttctaat 1500 ttaatattgt ctagatgtat tgaaccataa gtttagttgg tctcaggaat tgatttttaa 1560 tgaaataatg gtcattaggg gtgagt 1586
SEQ ID NO:6
S. rebaudiana
atggatgcaa tggcaactac tgagaaaaag cctcatgtga tcttcattcc atttcctgca 60 caatctcaca taaaggcaat gctaaagtta gcacaactat tacaccataa gggattacag 120 ataactttcg tgaataccga cttcatccat aatcaatttc tggaatctag tggccctcat 180 tgtttggacg gagccccagg gtttagattc gaaacaattc ctgacggtgt ttcacattcc 240 ccagaggcct ccatcccaat aagagagagt ttactgaggt caatagaaac caactttttg 300 gatcgtttca ttgacttggt cacaaaactt ccagacccac caacttgcat aatctctgat 360 ggctttctgt cagtgtttac tatcgacgct gccaaaaagt tgggtatccc agttatgatg 420 tactggactc ttgctgcatg cggtttcatg ggtttctatc acatccattc tcttatcgaa 480 aagggttttg ctccactgaa agatgcatca tacttaacca acggctacct ggatactgtt 540 attgactggg taccaggtat ggaaggtata agacttaaag attttccttt ggattggtct 600 acagacctta atgataaagt attgatgttt actacagaag ctccacaaag atctcataag 660 gtttcacatc atatctttca cacctttgat gaattggaac catcaatcat caaaaccttg 720 tctctaagat acaatcatat ctacactatt ggtccattac aattacttct agatcaaatt 780 cctgaagaga aaaagcaaac tggtattaca tccttacacg gctactcttt agtgaaagag 840 gaaccagaat gttttcaatg gctacaaagt aaagagccta attctgtggt ctacgtcaac 900 ttcggaagta caacagtcat gtccttggaa gatatgactg aatttggttg gggccttgct 960 aattcaaatc attactttct atggattatc aggtccaatt tggtaatagg ggaaaacgcc 1020 gtattacctc cagaattgga ggaacacatc aaaaagagag gtttcattgc ttcctggtgt 1080 tctcaggaaa aggtattgaa acatccttct gttggtggtt tccttactca ttgcggttgg 1140 ggctctacaa tcgaatcact aagtgcagga gttccaatga tttgttggcc atattcatgg 1200 gaccaactta caaattgtag gtatatctgt aaagagtggg aagttggatt agaaatggga 1260 acaaaggtta aacgtgatga agtgaaaaga ttggttcagg agttgatggg ggaaggtggc 1320 cacaagatga gaaacaaggc caaagattgg aaggaaaaag ccagaattgc tattgctcct 1380 aacgggtcat cctctctaaa cattgataag atggtcaaag agattacagt cttagccaga 1440 aactaa 1446
SEQ ID NO:7
S. rebaudiana
MDAMATTEKK PHVIFIPFPA QSHIKAMLKL AQLLHHKGLQ ITFVNTDFIH NQFLESSGPH 60
CLDGAPGFRF ETIPDGVSHS PEASIPIRES LLRSIETNFL DRFIDLVTKL PDPPTCIISD 120
GFLSVFTIDA AKKLGI PVMM YWTLAACGFM GFYHIHSLIE KGFAPLKDAS YLTNGYLDTV 180
IDWVPGMEGI RLKDFPLDWS TDLNDKVLMF TTEAPQRSHK VSHHIFHTFD ELEPSIIKTL 240
SLRYNHIYTI GPLQLLLDQI PEEKKQTGIT SLHGYSLVKE EPECFQWLQS KEPNSWYVN 300
FGSTTVMSLE DMTEFGWGLA NSNHYFLWII RSNLVIGENA VLPPELEEHI KKRGFIASWC 360
SQEKVLKHPS VGGFLTHCGW GSTIESLSAG VPMICWPYSW DQLTNCRYIC KEWEVGLEMG 420
TKVKRDEVKR LVQELMGEGG HKMRNKAKDW KEKARIAIAP NGSSSLNIDK MVKEITVLAR 480
N 481
SEQ ID NO:8
S. rebaudiana
atggaaaaca agaccgaaac aacagttaga cgtaggcgta gaatcattct gtttccagta 60 ccttttcaag ggcacatcaa tccaatacta caactagcca acgttttgta ctctaaaggt 120 ttttctatta caatctttca caccaatttc aacaaaccaa aaacatccaa ttacccacat 180 ttcacattca gattcatact tgataatgat ccacaagatg aacgtatttc aaacttacct 240 acccacggtc ctttagctgg aatgagaatt ccaatcatca atgaacatgg tgccgatgag 300 cttagaagag aattagagtt acttatgttg gcatccgaag aggacgagga agtctcttgt 360 ctgattactg acgctctatg gtactttgcc caatctgtgg ctgatagttt gaatttgagg 420 agattggtac taatgacatc cagtctgttt aactttcacg ctcatgttag tttaccacaa 480 tttgacgaat tgggatactt ggaccctgat gacaagacta ggttagagga acaggcctct 540 ggttttccta tgttgaaagt caaagatatc aagtctgcct attctaattg gcaaatcttg 600 aaagagatct taggaaagat gatcaaacag acaaaggctt catctggagt gatttggaac 660 agtttcaaag agttagaaga gtctgaattg gagactgtaa tcagagaaat tccagcacct 720 tcattcctga taccattacc aaaacatttg actgcttcct cttcctcttt gttggatcat 780 gacagaacag tttttcaatg gttggaccaa caaccaccta gttctgtttt gtacgtgtca 840 tttggtagta cttctgaagt cgatgaaaag gacttccttg aaatcgcaag aggcttagtc 900 gatagtaagc agtcattcct ttgggtcgtg cgtccaggtt tcgtgaaagg ctcaacatgg 960 gtcgaaccac ttccagatgg ttttctaggc gaaagaggta gaatagtcaa atgggttcct 1020 caacaggaag ttttagctca tggcgctatt ggggcattct ggactcattc cggatggaat 1080 tcaactttag aatcagtatg cgaaggggta cctatgatct tttcagattt tggtcttgat 1140 caaccactga acgcaagata catgtctgat gttttgaaag tgggtgtata tctagaaaat 1200 ggctgggaaa ggggtgaaat agctaatgca ataagacgtg ttatggttga tgaagagggg 1260 gagtatatca gacaaaacgc aagagtgctg aagcaaaagg ccgacgtttc tctaatgaag 1320 ggaggctctt catacgaatc cttagaatct cttgtttcct acatttcatc actgtaa 1377
SEQ ID NO:9
S. rebaudiana
MENKTETTVR RRRRIILFPV PFQGHINPIL QLANVLYSKG FSITIFHTNF NKPKTSNYPH 60
FTFRFILDND PQDERISNLP THGPLAGMRI PI INEHGADE LRRELELLML ASEEDEEVSC 120 LITDALWYFA QSVADSLNLR RLVLMTSSLF NFHAHVSLPQ FDELGYLDPD DKTRLEEQAS 180
GFPMLKVKDI KSAYSNWQIL KEILGKMIKQ TKASSGVIWN SFKELEESEL ETVIREIPAP 240
SFLIPLPKHL TASSSSLLDH DRTVFQWLDQ QPPSSVLYVS FGSTSEVDEK DFLEIARGLV 300
DSKQSFLWW RPGFVKGSTW VEPLPDGFLG ERGRIVKWVP QQEVLAHGAI GAFWTHSGWN 360
STLESVCEGV PMI FSDFGLD QPLNARYMSD VLKVGVYLEN GWERGEIANA IRRVMVDEEG 420
EYIRQNARVL KQKADVSLMK GGSSYESLES LVSYISSL 458
SEQ ID NO:10
atggctacat ctgattctat tgttgatgac aggaagcagt tgcatgtggc tactttccct 60 tggcttgctt tcggtcatat actgccttac ctacaactat caaaactgat agctgaaaaa 120 ggacataaag tgtcattcct ttcaacaact agaaacattc aaagattatc ttcccacata 180 tcaccattga ttaacgtcgt tcaattgaca cttccaagag tacaggaatt accagaagat 240 gctgaagcta caacagatgt gcatcctgaa gatatccctt acttgaaaaa ggcatccgat 300 ggattacagc ctgaggtcac tagattcctt gagcaacaca gtccagattg gatcatatac 360 gactacactc actattggtt gccttcaatt gcagcatcac taggcatttc tagggcacat 420 ttcagtgtaa ccacaccttg ggccattgct tacatgggtc catccgctga tgctatgatt 480 aacggcagtg atggtagaac taccgttgaa gatttgacaa ccccaccaaa gtggtttcca 540 tttccaacta aagtctgttg gagaaaacac gacttagcaa gactggttcc atacaaggca 600 ccaggaatct cagacggcta tagaatgggt ttagtcctta aagggtctga ctgcctattg 660 tctaagtgtt accatgagtt tgggacacaa tggctaccac ttttggaaac attacaccaa 720 gttcctgtcg taccagttgg tctattacct ccagaaatcc ctggtgatga gaaggacgag 780 acttgggttt caatcaaaaa gtggttagac gggaagcaaa aaggctcagt ggtatatgtg 840 gcactgggtt ccgaagtttt agtatctcaa acagaagttg tggaacttgc cttaggtttg 900 gaactatctg gattgccatt tgtctgggcc tacagaaaac caaaaggccc tgcaaagtcc 960 gattcagttg aattgccaga cggctttgtc gagagaacta gagatagagg gttggtatgg 1020 acttcatggg ctccacaatt gagaatcctg agtcacgaat ctgtgtgcgg tttcctaaca 1080 cattgtggtt ctggttctat agttgaagga ctgatgtttg gtcatccact tatcatgttg 1140 ccaatctttg gtgaccagcc tttgaatgca cgtctgttag aagataaaca agttggaatt 1200 gaaatcccac gtaatgagga agatggatgt ttaaccaagg agtctgtggc cagatcatta 1260 cgttccgttg tcgttgaaaa ggaaggcgaa atctacaagg ccaatgcccg tgaactttca 1320 aagatctaca atgacacaaa agtagagaag gaatatgttt ctcaatttgt agattaccta 1380 gagaaaaacg ctagagccgt agctattgat catgaatcct aa 1422
SEQ ID NO:1 1
S. rebaudiana
MATSDSIVDD RKQLHVATFP WLAFGHILPY LQLSKLIAEK GHKVSFLSTT RNIQRLSSHI 60
SPLINWQLT LPRVQELPED AEATTDVHPE DIPYLKKASD GLQPEVTRFL EQHSPDWIIY 120
DYTHYWLPSI AASLGISRAH FSVTTPWAIA YMGPSADAMI NGSDGRTTVE DLTTPPKWFP 180
FPTKVCWRKH DLARLVPYKA PGISDGYRMG LVLKGSDCLL SKCYHEFGTQ WLPLLETLHQ 240
VPWPVGLLP PEI PGDEKDE TWVSIKKWLD GKQKGSWYV ALGSEVLVSQ TEWELALGL 300
ELSGLPFVWA YRKPKGPAKS DSVELPDGFV ERTRDRGLVW TSWAPQLRIL SHESVCGFLT 360
HCGSGSIVEG LMFGHPLIML PI FGDQPLNA RLLEDKQVGI EIPRNEEDGC LTKESVARSL 420
RSVWEKEGE IYKANARELS KIYNDTKVEK EYVSQFVDYL EKNARAVAID HES 473
SEQ ID NO:12
atggctactt ctgattccat cgttgacgat agaaagcaat tgcatgttgc tacttttcca 60 tggttggctt tcggtcatat tttgccatac ttgcaattgt ccaagttgat tgctgaaaag 120 ggtcacaagg tttcattctt gtctaccacc agaaacatcc aaagattgtc ctctcatatc 180 tccccattga tcaacgttgt tcaattgact ttgccaagag tccaagaatt gccagaagat 240 gctgaagcta ctactgatgt tcatccagaa gatatccctt acttgaaaaa ggcttccgat 300 ggtttacaac cagaagttac tagattcttg gaacaacatt ccccagattg gatcatctac 360 gattatactc attactggtt gccatccatt gctgcttcat tgggtatttc tagagcccat 420 ttctctgtta ctactccatg ggctattgct tatatgggtc catctgctga tgctatgatt 480 aacggttctg atggtagaac taccgttgaa gatttgacta ctccaccaaa gtggtttcca 540 tttccaacaa aagtctgttg gagaaaacac gatttggcta gattggttcc atacaaagct 600 ccaggtattt ctgatggtta cagaatgggt atggttttga aaggttccga ttgcttgttg 660 tctaagtgct atcatgaatt cggtactcaa tggttgcctt tgttggaaac attgcatcaa 720 gttccagttg ttccagtagg tttgttgcca ccagaaattc caggtgacga aaaagacgaa 780 acttgggttt ccatcaaaaa gtggttggat ggtaagcaaa agggttctgt tgtttatgtt 840 gctttgggtt ccgaagcttt ggtttctcaa accgaagttg ttgaattggc tttgggtttg 900 gaattgtctg gtttgccatt tgtttgggct tacagaaaac ctaaaggtcc agctaagtct 960 gattctgttg aattgccaga tggtttcgtt gaaagaacta gagatagagg tttggtttgg 1020 acttcttggg ctccacaatt gagaattttg tctcatgaat ccgtctgtgg tttcttgact 1080 cattgtggtt ctggttctat cgttgaaggt ttgatgtttg gtcacccatt gattatgttg 1140 ccaatctttg gtgaccaacc attgaacgct agattattgg aagataagca agtcggtatc 1200 gaaatcccaa gaaatgaaga agatggttgc ttgaccaaag aatctgttgc tagatctttg 1260 agatccgttg tcgttgaaaa agaaggtgaa atctacaagg ctaacgctag agaattgtcc 1320 aagatctaca acgataccaa ggtcgaaaaa gaatacgttt cccaattcgt tgactacttg 1380 gaaaagaatg ctagagctgt tgccattgat catgaatctt ga 1422
SEQ ID NO:13
MATSDSIVDD RKQLHVATFP WLAFGHILPY LQLSKLIAEK GHKVSFLSTT RNIQRLSSHI 60
SPLINWQLT LPRVQELPED AEATTDVHPE DIPYLKKASD GLQPEVTRFL EQHSPDWIIY 120
DYTHYWLPSI AASLGISRAH FSVTTPWAIA YMGPSADAMI NGSDGRTTVE DLTTPPKWFP 180
FPTKVCWRKH DLARLVPYKA PGISDGYRMG MVLKGSDCLL SKCYHEFGTQ WLPLLETLHQ 240
VPWPVGLLP PEI PGDEKDE TWVSIKKWLD GKQKGSWYV ALGSEALVSQ TEWELALGL 300
ELSGLPFVWA YRKPKGPAKS DSVELPDGFV ERTRDRGLVW TSWAPQLRIL SHESVCGFLT 360
HCGSGSIVEG LMFGHPLIML PIFGDQPLNA RLLEDKQVGI EIPRNEEDGC LTKESVARSL 420
RSVWEKEGE IYKANARELS KIYNDTKVEK EYVSQFVDYL EKNARAVAID HES 473
SEQ ID NO:14
O. sativa
atggactccg gctactcctc ctcctacgcc gccgccgccg ggatgcacgt cgtgatctgc 60 ccgtggctcg ccttcggcca cctgctcccg tgcctcgacc tcgcccagcg cctcgcgtcg 120 cggggccacc gcgtgtcgtt cgtctccacg ccgcggaaca tatcccgcct cccgccggtg 180 cgccccgcgc tcgcgccgct cgtcgccttc gtggcgctgc cgctcccgcg cgtcgagggg 240 ctccccgacg gcgccgagtc caccaacgac gtcccccacg acaggccgga catggtcgag 300 ctccaccgga gggccttcga cgggctcgcc gcgcccttct cggagttctt gggcaccgcg 360 tgcgccgact gggtcatcgt cgacgtcttc caccactggg ccgcagccgc cgctctcgag 420 cacaaggtgc catgtgcaat gatgttgttg ggctctgcac atatgatcgc ttccatagca 480 gacagacggc tcgagcgcgc ggagacagag tcgcctgcgg ctgccgggca gggacgccca 540 gcggcggcgc caacgttcga ggtggcgagg atgaagttga tacgaaccaa aggctcatcg 600 ggaatgtccc tcgccgagcg cttctccttg acgctctcga ggagcagcct cgtcgtcggg 660 cggagctgcg tggagttcga gccggagacc gtcccgctcc tgtcgacgct ccgcggtaag 720 cctattacct tccttggcct tatgccgccg ttgcatgaag gccgccgcga ggacggcgag 780 gatgccaccg tccgctggct cgacgcgcag ccggccaagt ccgtcgtgta cgtcgcgcta 840 ggcagcgagg tgccactggg agtggagaag gtccacgagc tcgcgctcgg gctggagctc 900 gccgggacgc gcttcctctg ggctcttagg aagcccactg gcgtctccga cgccgacctc 960 ctccccgccg gcttcgagga gcgcacgcgc ggccgcggcg tcgtggcgac gagatgggtt 1020 cctcagatga gcatactggc gcacgccgcc gtgggcgcgt tcctgaccca ctgcggctgg 1080 aactcgacca tcgaggggct catgttcggc cacccgctta tcatgctgcc gatcttcggc 1140 gaccagggac cgaacgcgcg gctaatcgag gcgaagaacg ccggattgca ggtggcaaga 1200 aacgacggcg atggatcgtt cgaccgagaa ggcgtcgcgg cggcgattcg tgcagtcgcg 1260 gtggaggaag aaagcagcaa agtgtttcaa gccaaagcca agaagctgca ggagatcgtc 1320 gcggacatgg cctgccatga gaggtacatc gacggattca ttcagcaatt gagatcttac 1380 aaggattga 1389
SEQ ID NO:15
O. sativa
atggatagtg gctactcctc atcttatgct gctgccgctg gtatgcacgt tgtgatctgc 60 ccttggttgg cctttggtca cctgttacca tgtctggatt tagcccaaag actggcctca 120 agaggccata gagtatcatt tgtgtctact cctagaaata tctctcgttt accaccagtc 180 agacctgctc tagctcctct agttgcattc gttgctcttc cacttccaag agtagaagga 240 ttgccagacg gcgctgaatc tactaatgac gtaccacatg atagacctga catggtcgaa 300 ttgcatagaa gagcctttga tggattggca gctccatttt ctgagttcct gggcacagca 360 tgtgcagact gggttatagt cgatgtattt catcactggg ctgctgcagc cgcattggaa 420 cataaggtgc cttgtgctat gatgttgtta gggtcagcac acatgatcgc atccatagct 480 gatagaagat tggaaagagc tgaaacagaa tccccagccg cagcaggaca aggtaggcca 540 gctgccgccc caacctttga agtggctaga atgaaattga ttcgtactaa aggtagttca 600 gggatgagtc ttgctgaaag gttttctctg acattatcta gatcatcatt agttgtaggt 660 agatcctgcg tcgagttcga acctgaaaca gtacctttac tatctacttt gagaggcaaa 720 cctattactt tccttggtct aatgcctcca ttacatgaag gaaggagaga agatggtgaa 780 gatgctactg ttaggtggtt agatgcccaa cctgctaagt ctgttgttta cgttgcattg 840 ggttctgagg taccactagg ggtggaaaag gtgcatgaat tagcattagg acttgagctg 900 gccggaacaa gattcctttg ggctttgaga aaaccaaccg gtgtttctga cgccgacttg 960 ctaccagctg ggttcgaaga gagaacaaga ggccgtggtg tcgttgctac tagatgggtc 1020 ccacaaatga gtattctagc tcatgcagct gtaggggcct ttctaaccca ttgcggttgg 1080 aactcaacaa tagaaggact gatgtttggt catccactta ttatgttacc aatctttggc 1140 gatcagggac ctaacgcaag attgattgag gcaaagaacg caggtctgca ggttgcacgt 1200 aatgatggtg atggttcctt tgatagagaa ggcgttgcag ctgccatcag agcagtcgcc 1260 gttgaggaag agtcatctaa agttttccaa gctaaggcca aaaaattaca agagattgtg 1320 gctgacatgg cttgtcacga aagatacatc gatggtttca tccaacaatt gagaagttat 1380 aaagactaa 1389
SEQ ID NO:16
O. sativa
MDSGYSSSYA AAAGMHWIC PWLAFGHLLP CLDLAQRLAS RGHRVSFVST PRNISRLPPV 60
RPALAPLVAF VALPLPRVEG LPDGAESTND VPHDRPDMVE LHRRAFDGLA APFSEFLGTA 120
CADWVIVDVF HHWAAAAALE HKVPCAMMLL GSAHMIASIA DRRLERAETE SPAAAGQGRP 180
AAAPTFEVAR MKLIRTKGSS GMSLAERFSL TLSRSSLVVG RSCVEFEPET VPLLSTLRGK 240
PITFLGLMPP LHEGRREDGE DATVRWLDAQ PAKSWYVAL GSEVPLGVEK VHELALGLEL 300
AGTRFLWALR KPTGVSDADL LPAGFEERTR GRGWATRWV PQMSILAHAA VGAFLTHCGW 360
NSTIEGLMFG HPLIMLPIFG DQGPNARLIE AKNAGLQVAR NDGDGSFDRE GVAAAIRAVA 420
VEEESSKVFQ AKAKKLQEIV ADMACHERYI DGFIQQLRSY KD 462
SEQ ID NO:17
MDSGYSSSYA AAAGMHWIC PWLAFGHLLP CLDLAQRLAS RGHRVSFVST PRNISRLPPV 60
RPALAPLVAF VALPLPRVEG LPDGAESTND VPHDRPDMVE LHRRAFDGLA APFSEFLGTA 120
CADWVIVDVF HHWAAAAALE HKVPCAMMLL GSAHMIASIA DRRLERAETE SPAAAGQGRP 180
AAAPTFEVAR MKLIRTKGSS GMSLAERFSL TLSRSSLVVG RSCVEFEPET VPLLSTLRGK 240
PITFLGLLPP EIPGDEKDET WVSIKKWLDG KQKGSWYVA LGSEALVSQT EVVELALGLE 300
LSGLPFVWAY RKPKGPAKSD SVELPDGFVE RTRDRGLVWT SWAPQLRILS HESVCGFLTH 360
CGSGSIVEGL MFGHPLIMLP IFGDQPLNAR LLEDKQVGIE IARNDGDGSF DREGVAAAIR 420
AVAVEEESSK VFQAKAKKLQ EIVADMACHE RYIDGFIQQL RSYKD 465
SEQ ID NO:18
MATSDSIVDD RKQLHVATFP WLAFGHILPY LQLSKLIAEK GHKVSFLSTT RNIQRLSSHI 60
SPLINWQLT LPRVQELPED AEATTDVHPE DIPYLKKASD GLQPEVTRFL EQHSPDWIIY 120
DYTHYWLPSI AASLGISRAH FSVTTPWAIA YMGPSADAMI NGSDGRTTVE DLTTPPKWFP 180
FPTKVCWRKH DLARLVPYKA PGISDGYRMG MVLKGSDCLL SKCYHEFGTQ WLPLLETLHQ 240
VPWPVGLMP PLHEGRREDG EDATVRWLDA QPAKSVVYVA LGSEVPLGVE KVHELALGLE 300
LAGTRFLWAL RKPTGVSDAD LLPAGFEERT RGRGVVATRW VPQMSILAHA AVGAFLTHCG 360
WNSTIEGLMF GHPLIMLPIF GDQGPNARLI EAKNAGLQVP RNEEDGCLTK ESVARSLRSV 420
WEKEGEIYK ANARELSKIY NDTKVEKEYV SQFVDYLEKN ARAVAIDHES 470 SEQ ID NO:19
Synechococcus sp.
atggctttgg taaacccaac cgctcttttc tatggtacct ctatcagaac aagacctaca 60 aacttactaa atccaactca aaagctaaga ccagtttcat catcttcctt accttctttc 120 tcatcagtta gtgcgattct tactgaaaaa catcaatcta atccttctga gaacaacaat 180 ttgcaaactc atctagaaac tcctttcaac tttgatagtt atatgttgga aaaagtcaac 240 atggttaacg aggcgcttga tgcatctgtc ccactaaaag acccaatcaa aatccatgaa 300 tccatgagat actctttatt ggcaggcggt aagagaatca gaccaatgat gtgtattgca 360 gcctgcgaaa tagtcggagg taatatcctt aacgccatgc cagccgcatg tgccgtggaa 420 atgattcata ctatgtcttt ggtgcatgac gatcttccat gtatggataa tgatgacttc 480 agaagaggta aacctatttc acacaaggtc tacggggagg aaatggcagt attgaccggc 540 gatgctttac taagtttatc tttcgaacat atagctactg ctacaaaggg tgtatcaaag 600 gatagaatcg tcagagctat aggggagttg gcccgttcag ttggctccga aggtttagtg 660 gctggacaag ttgtagatat cttgtcagag ggtgctgatg ttggattaga tcacctagaa 720 tacattcaca tccacaaaac agcaatgttg cttgagtcct cagtagttat tggcgctatc 780 atgggaggag gatctgatca gcagatcgaa aagttgagaa aattcgctag atctattggt 840 ctactattcc aagttgtgga tgacattttg gatgttacaa aatctaccga agagttgggg 900 aaaacagctg gtaaggattt gttgacagat aagacaactt acccaaagtt gttaggtata 960 gaaaagtcca gagaatttgc cgaaaaactt aacaaggaag cacaagagca attaagtggc 1020 tttgatagac gtaaggcagc tcctttgatc gcgttagcca actacaatgc gtaccgtcaa 1080 aattga 1086
SEQ ID NO:20
Synechococcus sp.
MALVNPTALF YGTSIRTRPT NLLNPTQKLR PVSSSSLPSF SSVSAILTEK HQSNPSENNN 60
LQTHLETPFN FDSYMLEKVN MVNEALDASV PLKDPIKIHE SMRYSLLAGG KRIRPMMCIA 120
ACEIVGGNIL NAMPAACAVE MIHTMSLVHD DLPCMDNDDF RRGKPISHKV YGEEMAVLTG 180
DALLSLSFEH IATATKGVSK DRIVRAIGEL ARSVGSEGLV AGQVVDILSE GADVGLDHLE 240
YIHIHKTAML LESSWIGAI MGGGSDQQIE KLRKFARSIG LLFQWDDIL DVTKSTEELG 300
KTAGKDLLTD KTTYPKLLGI EKSREFAEKL NKEAQEQLSG FDRRKAAPLI ALANYNAYRQ 360
N 361
SEQ ID NO:21
atggctgagc aacaaatatc taacttgctg tctatgtttg atgcttcaca tgctagtcag 60 aaattagaaa ttactgtcca aatgatggac acataccatt acagagaaac gcctccagat 120 tcctcatctt ctgaaggcgg ttcattgtct agatacgacg agagaagagt ctctttgcct 180 ctcagtcata atgctgcctc tccagatatt gtatcacaac tatgtttttc cactgcaatg 240 tcttcagagt tgaatcacag atggaaatct caaagattaa aggtggccga ttctccttac 300 aactatatcc taacattacc atcaaaagga attagaggtg cctttatcga ttccctgaac 360 gtatggttgg aggttccaga ggatgaaaca tcagtcatca aggaagttat tggtatgctc 420 cacaactctt cattaatcat tgatgacttc caagataatt ctccacttag aagaggaaag 480 ccatctaccc atacagtctt cggccctgcc caggctatca atactgctac ttacgttata 540 gttaaagcaa tcgaaaagat acaagacata gtgggacacg atgcattggc agatgttacg 600 ggtactatta caactatttt ccaaggtcag gccatggact tgtggtggac agcaaatgca 660 atcgttccat caatacagga atacttactt atggtaaacg ataaaaccgg tgctctcttt 720 agactgagtt tggagttgtt agctctgaat tccgaagcca gtatttctga ctctgcttta 780 gaaagtttat ctagtgctgt ttccttgcta ggtcaatact tccaaatcag agacgactat 840 atgaacttga tcgataacaa gtatacagat cagaaaggct tctgcgaaga tcttgatgaa 900 ggcaagtact cactaacact tattcatgcc ctccaaactg attcatccga tctactgacc 960 aacatccttt caatgagaag agtgcaagga aagttaacgg cacaaaagag atgttggttc 1020 tggaaatga 1029
SEQ ID NO:22
Gibberella fujikuroi
MAEQQISNLL SMFDASHASQ KLEITVQMMD TYHYRETPPD SSSSEGGSLS RYDERRVSLP 60 LSHNAASPDI VSQLCFSTAM SSELNHRWKS QRLKVADSPY NYILTLPSKG IRGAFIDSLN 120
VWLEVPEDET SVIKEVIGML HNSSLIIDDF QDNSPLRRGK PSTHTVFGPA QAINTATYVI 180
VKAIEKIQDI VGHDALADVT GTITTIFQGQ AMDLWWTANA IVPSIQEYLL MVNDKTGALF 240
RLSLELLALN SEASISDSAL ESLSSAVSLL GQYFQIRDDY MNLIDNKYTD QKGFCEDLDE 300
GKYSLTLIHA LQTDSSDLLT NILSMRRVQG KLTAQKRCWF WK 342
SEQ ID NO:23
atggaaaaga ctaaggagaa agcagaacgt atcttgctgg agccatacag atacttatta 60 caactaccag gaaagcaagt ccgttctaaa ctatcacaag cgttcaatca ctggttaaaa 120 gttcctgaag ataagttaca aatcattatt gaagtcacag aaatgctaca caatgcttct 180 ttactgatcg atgatataga ggattcttcc aaactgagaa gaggttttcc tgtcgctcat 240 tccatatacg gggtaccaag tgtaatcaac tcagctaatt acgtctactt cttgggattg 300 gaaaaagtat tgacattaga tcatccagac gctgtaaagc tattcaccag acaacttctt 360 gaattgcatc aaggtcaagg tttggatatc tattggagag acacttatac ttgcccaaca 420 gaagaggagt acaaagcaat ggttctacaa aagactggcg gtttgttcgg acttgccgtt 480 ggtctgatgc aacttttctc tgattacaag gaggacttaa agcctctgtt ggataccttg 540 ggcttgtttt tccagattag agatgactac gctaacttac attcaaagga atattcagaa 600 aacaaatcat tctgtgaaga tttgactgaa gggaagttta gttttccaac aatccacgcc 660 atttggtcaa gaccagaatc tactcaagtg caaaacattc tgcgtcagag aacagagaat 720 attgacatca aaaagtattg tgttcagtac ttggaagatg ttggttcttt tgcttacaca 780 agacatacac ttagagaatt agaggcaaaa gcatacaagc aaatagaagc ctgtggaggc 840 aatccttctc tagtggcatt ggttaaacat ttgtccaaaa tgttcaccga ggaaaacaag 900 taa 903
SEQ ID NO:24
Mus musculus
MEKTKEKAER ILLEPYRYLL QLPGKQVRSK LSQAFNHWLK VPEDKLQIII EVTEMLHNAS 60
LLIDDIEDSS KLRRGFPVAH SIYGVPSVIN SANYVYFLGL EKVLTLDHPD AVKLFTRQLL 120
ELHQGQGLDI YWRDTYTCPT EEEYKAMVLQ KTGGLFGLAV GLMQLFSDYK EDLKPLLDTL 180
GLFFQIRDDY ANLHSKEYSE NKSFCEDLTE GKFSFPTIHA IWSRPESTQV QNILRQRTEN 240
IDIKKYCVQY LEDVGSFAYT RHTLRELEAK AYKQIEACGG NPSLVALVKH LSKMFTEENK 300
SEQ ID NO:25
atggcaagat tctattttct taacgcacta ttgatggtta tctcattaca atcaactaca 60 gccttcactc cagctaaact tgcttatcca acaacaacaa cagctctaaa tgtcgcctcc 120 gccgaaactt ctttcagtct agatgaatac ttggcctcta agataggacc tatagagtct 180 gccttggaag catcagtcaa atccagaatt ccacagaccg ataagatctg cgaatctatg 240 gcctactctt tgatggcagg aggcaagaga attagaccag tgttgtgtat cgctgcatgt 300 gagatgttcg gtggatccca agatgtcgct atgcctactg ctgtggcatt agaaatgata 360 cacacaatgt ctttgattca tgatgatttg ccatccatgg ataacgatga cttgagaaga 420 ggtaaaccaa caaaccatgt cgttttcggc gaagatgtag ctattcttgc aggtgactct 480 ttattgtcaa cttccttcga gcacgtcgct agagaaacaa aaggagtgtc agcagaaaag 540 atcgtggatg ttatcgctag attaggcaaa tctgttggtg ccgagggcct tgctggcggt 600 caagttatgg acttagaatg tgaagctaaa ccaggtacca cattagacga cttgaaatgg 660 attcatatcc ataaaaccgc tacattgtta caagttgctg tagcttctgg tgcagttcta 720 ggtggtgcaa ctcctgaaga ggttgctgca tgcgagttgt ttgctatgaa tataggtctt 780 gcctttcaag ttgccgacga tatccttgat gtaaccgctt catcagaaga tttgggtaaa 840 actgcaggca aagatgaagc tactgataag acaacttacc caaagttatt aggattagaa 900 gagagtaagg catacgcaag acaactaatc gatgaagcca aggaaagttt ggctcctttt 960 ggagatagag ctgccccttt attggccatt gcagatttca ttattgatag aaagaattga 1020
SEQ ID NO:26
Thalassiosira pseudonana
MARFYFLNAL LMVISLQSTT AFTPAKLAYP TTTTALNVAS AETSFSLDEY LASKIGPIES 60
ALEASVKSRI PQTDKICESM AYSLMAGGKR IRPVLCIAAC EMFGGSQDVA MPTAVALEMI 120 HTMSLIHDDL PSMDNDDLRR GKPTNHVVFG EDVAILAGDS LLSTSFEHVA RETKGVSAEK 180
IVDVIARLGK SVGAEGLAGG QVMDLECEAK PGTTLDDLKW IHIHKTATLL QVAVASGAVL 240
GGATPEEVAA CELFAMNIGL AFQVADDILD VTASSEDLGK TAGKDEATDK TTYPKLLGLE 300
ESKAYARQLI DEAKESLAPF GDRAAPLLAI ADFIIDRKN 339
SEQ ID NO:27
atgcacttag caccacgtag agtccctaga ggtagaagat caccacctga cagagttcct 60 gaaagacaag gtgccttggg tagaagacgt ggagctggct ctactggctg tgcccgtgct 120 gctgctggtg ttcaccgtag aagaggagga ggcgaggctg atccatcagc tgctgtgcat 180 agaggctggc aagccggtgg tggcaccggt ttgcctgatg aggtggtgtc taccgcagcc 240 gccttagaaa tgtttcatgc ttttgcttta atccatgatg atatcatgga tgatagtgca 300 actagaagag gctccccaac tgttcacaga gccctagctg atcgtttagg cgctgctctg 360 gacccagatc aggccggtca actaggagtt tctactgcta tcttggttgg agatctggct 420 ttgacatggt ccgatgaatt gttatacgct ccattgactc cacatagact ggcagcagta 480 ctaccattgg taacagctat gagagctgaa accgttcatg gccaatatct tgatataact 540 agtgctagaa gacctgggac cgatacttct cttgcattga gaatagccag atataagaca 600 gcagcttaca caatggaacg tccactgcac attggtgcag ccctggctgg ggcaagacca 660 gaactattag cagggctttc agcatacgcc ttgccagctg gagaagcctt ccaattggca 720 gatgacctgc taggcgtctt cggtgatcca agacgtacag ggaaacctga cctagatgat 780 cttagaggtg gaaagcatac tgtcttagtc gccttggcaa gagaacatgc cactccagaa 840 cagagacaca cattggatac attattgggt acaccaggtc ttgatagaca aggcgcttca 900 agactaagat gcgtattggt agcaactggt gcaagagccg aagccgaaag acttattaca 960 gagagaagag atcaagcatt aactgcattg aacgcattaa cactgccacc tcctttagct 1020 gaggcattag caagattgac attagggtct acagctcatc ctgcctaa 1068
SEQ ID NO:28
Streptomyces clavuligerus
MHLAPRRVPR GRRSPPDRVP ERQGALGRRR GAGSTGCARA AAGVHRRRGG GEADPSAAVH 60
RGWQAGGGTG LPDEWSTAA ALEMFHAFAL IHDDIMDDSA TRRGSPTVHR ALADRLGAAL 120
DPDQAGQLGV STAILVGDLA LTWSDELLYA PLTPHRLAAV LPLVTAMRAE TVHGQYLDIT 180
SARRPGTDTS LALRIARYKT AAYTMERPLH IGAALAGARP ELLAGLSAYA LPAGEAFQLA 240
DDLLGVFGDP RRTGKPDLDD LRGGKHTVLV ALAREHATPE QRHTLDTLLG TPGLDRQGAS 300
RLRCVLVATG ARAEAERLIT ERRDQALTAL NALTLPPPLA EALARLTLGS TAHPA 355
SEQ ID NO:29
atgtcatatt tcgataacta cttcaatgag atagttaatt ccgtgaacga catcattaag 60 tcttacatct ctggcgacgt accaaaacta tacgaagcct cctaccattt gtttacatca 120 ggaggaaaga gactaagacc attgatcctt acaatttctt ctgatctttt cggtggacag 180 agagaaagag catactatgc tggcgcagca atcgaagttt tgcacacatt cactttggtt 240 cacgatgata tcatggatca agataacatt cgtagaggtc ttcctactgt acatgtcaag 300 tatggcctac ctttggccat tttagctggt gacttattgc atgcaaaagc ctttcaattg 360 ttgactcagg cattgagagg tctaccatct gaaactatca tcaaggcgtt tgatatcttt 420 acaagatcta tcattatcat atcagaaggt caagctgtcg atatggaatt cgaagataga 480 attgatatca aggaacaaga gtatttggat atgatatctc gtaaaaccgc tgccttattc 540 tcagcttctt cttccattgg ggcgttgata gctggagcta atgataacga tgtgagatta 600 atgtccgatt tcggtacaaa tcttgggatc gcatttcaaa ttgtagatga tatacttggt 660 ttaacagctg atgaaaaaga gctaggaaaa cctgttttca gtgatatcag agaaggtaaa 720 aagaccatat tagtcattaa gactttagaa ttgtgtaagg aagacgagaa aaagattgtg 780 ttaaaagcgc taggcaacaa gtcagcatca aaggaagagt tgatgagttc tgctgacata 840 atcaaaaagt actcattgga ttacgcctac aacttagctg agaaatacta caaaaacgcc 900 atcgattctc taaatcaagt ttcaagtaaa agtgatattc cagggaaggc attgaaatat 960 cttgctgaat tcaccatcag aagacgtaag taa 993
SEQ ID NO:30
Sulfolobus acidocaldarius MSYFDNYFNE IVNSVNDIIK SYISGDVPKL YEASYHLFTS GGKRLRPLIL TISSDLFGGQ 60
RERAYYAGAA IEVLHTFTLV HDDIMDQDNI RRGLPTVHVK YGLPLAILAG DLLHAKAFQL 120
LTQALRGLPS ETIIKAFDIF TRSIIIISEG QAVDMEFEDR IDIKEQEYLD MISRKTAALF 180
SASSSIGALI AGANDNDVRL MSDFGTNLGI AFQIVDDILG LTADEKELGK PVFSDIREGK 240
KTILVIKTLE LCKEDEKKIV LKALGNKSAS KEELMSSADI IKKYSLDYAY NLAEKYYKNA 300
IDSLNQVSSK SDI PGKALKY LAEFTIRRRK 330
SEQ ID NO:31
atggtcgcac aaactttcaa cctggatacc tacttatccc aaagacaaca acaagttgaa 60 gaggccctaa gtgctgctct tgtgccagct tatcctgaga gaatatacga agctatgaga 120 tactccctcc tggcaggtgg caaaagatta agacctatct tatgtttagc tgcttgcgaa 180 ttggcaggtg gttctgttga acaagccatg ccaactgcgt gtgcacttga aatgatccat 240 acaatgtcac taattcatga tgacctgcca gccatggata acgatgattt cagaagagga 300 aagccaacta atcacaaggt gttcggggaa gatatagcca tcttagcggg tgatgcgctt 360 ttagcttacg cttttgaaca tattgcttct caaacaagag gagtaccacc tcaattggtg 420 ctacaagtta ttgctagaat cggacacgcc gttgctgcaa caggcctcgt tggaggccaa 480 gtcgtagacc ttgaatctga aggtaaagct atttccttag aaacattgga gtatattcac 540 tcacataaga ctggagcctt gctggaagca tcagttgtct caggcggtat tctcgcaggg 600 gcagatgaag agcttttggc cagattgtct cattacgcta gagatatagg cttggctttt 660 caaatcgtcg atgatatcct ggatgttact gctacatctg aacagttggg gaaaaccgct 720 ggtaaagacc aggcagccgc aaaggcaact tatccaagtc tattgggttt agaagcctct 780 agacagaaag cggaagagtt gattcaatct gctaaggaag ccttaagacc ttacggttca 840 caagcagagc cactcctagc gctggcagac ttcatcacac gtcgtcagca ttaa 894
SEQ ID NO:32
Synechococcus sp.
MVAQTFNLDT YLSQRQQQVE EALSAALVPA YPERIYEAMR YSLLAGGKRL RPILCLAACE 60
LAGGSVEQAM PTACALEMIH TMSLIHDDLP AMDNDDFRRG KPTNHKVFGE DIAILAGDAL 120
LAYAFEHIAS QTRGVPPQLV LQVIARIGHA VAATGLVGGQ WDLESEGKA ISLETLEYIH 180
SHKTGALLEA SWSGGILAG ADEELLARLS HYARDIGLAF QIVDDILDVT ATSEQLGKTA 240
GKDQAAAKAT YPSLLGLEAS RQKAEELIQS AKEALRPYGS QAEPLLALAD FITRRQH 297
SEQ ID NO:33
atgaaaaccg ggtttatctc accagcaaca gtatttcatc acagaatctc accagcgacc 60 actttcagac atcacttatc acctgctact acaaactcta caggcattgt cgccttaaga 120 gacatcaact tcagatgtaa agcagtttct aaagagtact ctgatctgtt gcagaaagat 180 gaggcttctt tcacaaaatg ggacgatgac aaggtgaaag atcatcttga taccaacaaa 240 aacttatacc caaatgatga gattaaggaa tttgttgaat cagtaaaggc tatgttcggt 300 agtatgaatg acggggagat aaacgtctct gcatacgata ctgcatgggt tgctttggtt 360 caagatgtcg atggatcagg tagtcctcag ttcccttctt ctttagaatg gattgccaac 420 aatcaattgt cagatggatc atggggagat catttgctgt tctcagctca cgatagaatc 480 atcaacacat tagcatgcgt tattgcactt acaagttgga atgttcatcc ttctaagtgt 540 gaaaaaggtt tgaattttct gagagaaaac atttgcaaat tagaagatga aaacgcagaa 600 catatgccaa ttggttttga agtaacattc ccatcactaa ttgatatcgc gaaaaagttg 660 aacattgaag tacctgagga tactccagca cttaaagaga tctacgcacg tagagatatc 720 aagttaacta agatcccaat ggaagttctt cacaaggtac ctactacttt gttacattct 780 ttggaaggaa tgcctgattt ggagtgggaa aaactgttaa agctacaatg taaagatggt 840 agtttcttgt tttccccatc tagtaccgca ttcgccctaa tgcaaacaaa agatgagaaa 900 tgcttacagt atctaacaaa tatcgtcact aagttcaacg gtggcgtgcc taatgtgtac 960 ccagtcgatt tgtttgaaca tatttgggtt gttgatagac tgcagagatt ggggattgcc 1020 agatacttca aatcagagat aaaagattgt gtagagtata tcaataagta ctggaccaaa 1080 aatggaattt gttgggctag aaatactcac gttcaagata tcgatgatac agccatggga 1140 ttcagagtgt tgagagcgca cggttatgac gtcactccag atgtttttag acaatttgaa 1200 aaagatggta aattcgtttg ctttgcaggg caatcaacac aagccgtgac aggaatgttt 1260 aacgtttaca gagcctctca aatgttgttc ccaggggaga gaattttgga agatgccaaa 1320 aagttctctt acaattactt aaaggaaaag caaagtacca acgaattgct ggataaatgg 1380 ataatcgcta aagatctacc tggtgaagtt ggttatgctc tggatatccc atggtatgct 1440 tccttaccaa gattggaaac tcgttattac cttgaacaat acggcggtga agatgatgtc 1500 tggataggca agacattata cagaatgggt tacgtgtcca ataacacata tctagaaatg 1560 gcaaagctgg attacaataa ctatgttgca gtccttcaat tagaatggta cacaatacaa 1620 caatggtacg tcgatattgg tatagagaag ttcgaatctg acaacatcaa gtcagtcctg 1680
SEQ ID NO:34
Stevia rebaudiana
MKTGFISPAT VFHHRISPAT TFRHHLSPAT TNSTGIVALR DINFRCKAVS KEYSDLLQKD 60
EASFTKWDDD KVKDHLDTNK NLYPNDEIKE FVESVKAMFG SMNDGEINVS AYDTAWVALV 120
QDVDGSGSPQ FPSSLEWIAN NQLSDGSWGD HLLFSAHDRI INTLACVIAL TSWNVHPSKC 180
EKGLNFLREN ICKLEDENAE HMPIGFEVTF PSLIDIAKKL NIEVPEDTPA LKEIYARRDI 240
KLTKIPMEVL HKVPTTLLHS LEGMPDLEWE KLLKLQCKDG SFLFSPSSTA FALMQTKDEK 300
CLQYLTNIVT KFNGGVPNVY PVDLFEHIWV VDRLQRLGIA RYFKSEIKDC VEYINKYWTK 360
NGICWARNTH VQDIDDTAMG FRVLRAHGYD VTPDVFRQFE KDGKFVCFAG QSTQAVTGMF 420
NVYRASQMLF PGERILEDAK KFSYNYLKEK QSTNELLDKW I IAKDLPGEV GYALDIPWYA 480
SLPRLETRYY LEQYGGEDDV WIGKTLYRMG YVSNNTYLEM AKLDYNNYVA VLQLEWYTIQ 540
QWYVDIGIEK FESDNIKSVL VSYYLAAASI FEPERSKERI AWAKTTILVD KITSIFDSSQ 600
SSKEDITAFI DKFRNKSSSK KHSINGEPWH EVMVALKKTL HGFALDALMT HSQDIHPQLH 660
QAWEMWLTKL QDGVDVTAEL MVQMINMTAG RWVSKELLTH PQYQRLSTVT NSVCHDITKL 720
HNFKENSTTV DSKVQELVQL VFSDTPDDLD QDMKQTFLTV MKTFYYKAWC DPNTINDHIS 780
KVFEIVI 787
SEQ ID NO:35
atgcctgatg cacacgatgc tccacctcca caaataagac agagaacact agtagatgag 60 gctacccaac tgctaactga gtccgcagaa gatgcatggg gtgaagtcag tgtgtcagaa 120 tacgaaacag caaggctagt tgcccatgct acatggttag gtggacacgc cacaagagtg 180 gccttccttc tggagagaca acacgaagac gggtcatggg gtccaccagg tggatatagg 240 ttagtcccta cattatctgc tgttcacgca ttattgacat gtcttgcctc tcctgctcag 300 gatcatggcg ttccacatga tagactttta agagctgttg acgcaggctt gactgccttg 360 agaagattgg ggacatctga ctccccacct gatactatag cagttgagct ggttatccca 420 tctttgctag agggcattca acacttactg gaccctgctc atcctcatag tagaccagcc 480 ttctctcaac atagaggctc tcttgtttgt cctggtggac tagatgggag aactctagga 540 gctttgagat cacacgccgc agcaggtaca ccagtaccag gaaaagtctg gcacgcttcc 600 gagactttgg gcttgagtac cgaagctgct tctcacttgc aaccagccca aggtataatc 660 ggtggctctg ctgctgccac agcaacatgg ctaaccaggg ttgcaccatc tcaacagtca 720 gattctgcca gaagatacct tgaggaatta caacacagat actctggccc agttccttcc 780 attaccccta tcacatactt cgaaagagca tggttattga acaattttgc agcagccggt 840 gttccttgtg aggctccagc tgctttgttg gattccttag aagcagcact tacaccacaa 900 ggtgctcctg ctggagcagg attgcctcca gatgctgatg atacagccgc tgtgttgctt 960 gcattggcaa cacatgggag aggtagaaga ccagaagtac tgatggatta caggactgac 1020 gggtatttcc aatgctttat tggggaaagg actccatcaa tttcaacaaa cgctcacgta 1080 ttggaaacat tagggcatca tgtggcccaa catccacaag atagagccag atacggatca 1140 gccatggata ccgcatcagc ttggctgctg gcagctcaaa agcaagatgg ctcttggtta 1200 gataaatggc atgcctcacc atactacgct actgtttgtt gcacacaagc cctagccgct 1260 catgcaagtc ctgcaactgc accagctaga cagagagctg tcagatgggt tttagccaca 1320 caaagatccg atggcggttg gggtctatgg cattcaactg ttgaagagac tgcttatgcc 1380 ttacagatct tggccccacc ttctggtggt ggcaatatcc cagtccaaca agcacttact 1440 agaggcagag caagattgtg tggagccttg ccactgactc ctttatggca tgataaggat 1500 ttgtatactc cagtaagagt agtcagagct gccagagctg ctgctctgta cactaccaga 1560 gatctattgt taccaccatt gtaa 1584
SEQ ID NO:36
Streptomyces clavuligerus MPDAHDAPPP QIRQRTLVDE ATQLLTESAE DAWGEVSVSE YETARLVAHA TWLGGHATRV 60
AFLLERQHED GSWGPPGGYR LVPTLSAVHA LLTCLASPAQ DHGVPHDRLL RAVDAGLTAL 120
RRLGTSDSPP DTIAVELVIP SLLEGIQHLL DPAHPHSRPA FSQHRGSLVC PGGLDGRTLG 180
ALRSHAAAGT PVPGKVWHAS ETLGLSTEAA SHLQPAQGII GGSAAATATW LTRVAPSQQS 240
DSARRYLEEL QHRYSGPVPS ITPITYFERA WLLNNFAAAG VPCEAPAALL DSLEAALTPQ 300
GAPAGAGLPP DADDTAAVLL ALATHGRGRR PEVLMDYRTD GYFQCFIGER TPSISTNAHV 360
LETLGHHVAQ HPQDRARYGS AMDTASAWLL AAQKQDGSWL DKWHASPYYA TVCCTQALAA 420
HASPATAPAR QRAVRWVLAT QRSDGGWGLW HSTVEETAYA LQILAPPSGG GNIPVQQALT 480
RGRARLCGAL PLTPLWHDKD LYTPVRVVRA ARAAALYTTR DLLLPPL 527
SEQ ID NO:37
atgaacgccc tatccgaaca cattttgtct gaattgagaa gattattgtc tgaaatgagt 60 gatggcggat ctgttggtcc atctgtgtat gatacggccc aggccctaag attccacggt 120 aacgtaacag gtagacaaga tgcatatgct tggttgatcg cccagcaaca agcagatgga 180 ggttggggct ctgccgactt tccactcttt agacatgctc caacatgggc tgcacttctc 240 gcattacaaa gagctgatcc acttcctggc gcagcagacg cagttcagac cgcaacaaga 300 ttcttgcaaa gacaaccaga tccatacgct catgccgttc ctgaggatgc ccctattggt 360 gctgaactga tcttgcctca gttttgtgga gaggctgctt ggttgttggg aggtgtggcc 420 ttccctagac acccagccct attaccatta agacaggctt gtttagtcaa actgggtgca 480 gtcgccatgt tgccttcagg acacccattg ctccactcct gggaggcatg gggtacttct 540 ccaacaacag cctgtccaga cgatgatggt tctataggta tctcaccagc agctacagcc 600 gcctggagag cccaggctgt gaccagaggc tcaactcctc aagtgggcag agctgacgca 660 tacttacaaa tggcttcaag agcaacgaga tcaggcatag aaggagtctt ccctaatgtt 720 tggcctataa acgtattcga accatgctgg tcactgtaca ctctccatct tgccggtctg 780 ttcgcccatc cagcactggc tgaggctgta agagttatcg ttgctcaact tgaagcaaga 840 ttgggagtgc atggcctcgg accagcttta cattttgctg ccgacgctga tgatactgca 900 gttgccttat gcgttctgca tttggctggc agagatcctg cagttgacgc attgagacat 960 tttgaaattg gtgagctctt tgttacattc ccaggagaga gaaatgctag tgtctctacg 1020 aacattcacg ctcttcatgc tttgagattg ttaggtaaac cagctgccgg agcaagtgca 1080 tacgtcgaag caaatagaaa tccacatggt ttgtgggaca acgaaaaatg gcacgtttca 1140 tggctttatc caactgcaca cgccgttgca gctctagctc aaggcaagcc tcaatggaga 1200 gatgaaagag cactagccgc tctactacaa gctcaaagag atgatggtgg ttggggagct 1260 ggtagaggat ccactttcga ggaaaccgcc tacgctcttt tcgctttaca cgttatggac 1320 ggatctgagg aagccacagg cagaagaaga atcgctcaag tcgtcgcaag agccttagaa 1380 tggatgctag ctagacatgc cgcacatgga ttaccacaaa caccactctg gattggtaag 1440 gaattgtact gtcctactag agtcgtaaga gtagctgagc tagctggcct gtggttagca 1500 ttaagatggg gtagaagagt attagctgaa ggtgctggtg ctgcacctta a 1551
SEQ ID NO:38
Bradyrhizobium japonicum
MNALSEHILS ELRRLLSEMS DGGSVGPSVY DTAQALRFHG NVTGRQDAYA WLIAQQQADG 60 GWGSADFPLF RHAPTWAALL ALQRADPLPG AADAVQTATR FLQRQPDPYA HAVPEDAPIG 120 AELILPQFCG EAAWLLGGVA FPRHPALLPL RQACLVKLGA VAMLPSGHPL LHSWEAWGTS 180 PTTACPDDDG SIGISPAATA AWRAQAVTRG STPQVGRADA YLQMASRATR SGIEGVFPNV 240 WPINVFEPCW SLYTLHLAGL FAHPALAEAV RVIVAQLEAR LGVHGLGPAL HFAADADDTA 300 VALCVLHLAG RDPAVDALRH FEIGELFVTF PGERNASVST NIHALHALRL LGKPAAGASA 360 YVEANRNPHG LWDNEKWHVS WLYPTAHAVA ALAQGKPQWR DERALAALLQ AQRDDGGWGA 420 GRGSTFEETA YALFALHVMD GSEEATGRRR IAQWARALE WMLARHAAHG LPQTPLWIGK 480 ELYCPTRVVR VAELAGLWLA LRWGRRVLAE GAGAAP 516
SEQ ID NO:39
Z. mays
atggttttgt cttcttcttg tactacagta ccacacttat cttcattagc tgtcgtgcaa 60 cttggtcctt ggagcagtag gattaaaaag aaaaccgata ctgttgcagt accagccgct 120 gcaggaaggt ggagaagggc cttggctaga gcacagcaca catcagaatc cgcagctgtc 180 gcaaagggca gcagtttgac ccctatagtg agaactgacg ctgagtcaag gagaacaaga 240 tggccaaccg atgacgatga cgccgaacct ttagtggatg agatcagggc aatgcttact 300 tccatgtctg atggtgacat ttccgtgagc gcatacgata cagcctgggt cggattggtt 360 ccaagattag acggcggtga aggtcctcaa tttccagcag ctgtgagatg gataagaaat 420 aaccagttgc ctgacggaag ttggggcgat gccgcattat tctctgccta tgacaggctt 480 atcaataccc ttgcctgcgt tgtaactttg acaaggtggt ccctagaacc agagatgaga 540 ggtagaggac tatctttttt gggtaggaac atgtggaaat tagcaactga agatgaagag 600 tcaatgccta ttggcttcga attagcattt ccatctttga tagagcttgc taagagccta 660 ggtgtccatg acttccctta tgatcaccag gccctacaag gaatctactc ttcaagagag 720 atcaaaatga agaggattcc aaaagaagtg atgcataccg ttccaacatc aatattgcac 780 agtttggagg gtatgcctgg cctagattgg gctaaactac ttaaactaca gagcagcgac 840 ggaagttttt tgttctcacc agctgccact gcatatgctt taatgaatac cggagatgac 900 aggtgtttta gctacatcga tagaacagta aagaaattca acggcggcgt ccctaatgtt 960 tatccagtgg atctatttga acatatttgg gccgttgata gacttgaaag attaggaatc 1020 tccaggtact tccaaaagga gatcgaacaa tgcatggatt atgtaaacag gcattggact 1080 gaggacggta tttgttgggc aaggaactct gatgtcaaag aggtggacga cacagctatg 1140 gcctttagac ttcttaggtt gcacggctac agcgtcagtc ctgatgtgtt taaaaacttc 1200 gaaaaggacg gtgaattttt cgcatttgtc ggacagtcta atcaagctgt taccggtatg 1260 tacaacttaa acagagcaag ccagatatcc ttcccaggcg aggatgtgct tcatagagct 1320 ggtgccttct catatgagtt cttgaggaga aaagaagcag agggagcttt gagggacaag 1380 tggatcattt ctaaagatct acctggtgaa gttgtgtata ctttggattt tccatggtac 1440 ggcaacttac ctagagtcga ggccagagac tacctagagc aatacggagg tggtgatgac 1500 gtttggattg gcaagacatt gtataggatg ccacttgtaa acaatgatgt atatttggaa 1560 ttggcaagaa tggatttcaa ccactgccag gctttgcatc agttagagtg gcaaggacta 1620 aaaagatggt atactgaaaa taggttgatg gactttggtg tcgcccaaga agatgccctt 1680 agagcttatt ttcttgcagc cgcatctgtt tacgagcctt gtagagctgc cgagaggctt 1740 gcatgggcta gagccgcaat actagctaac gccgtgagca cccacttaag aaatagccca 1800 tcattcagag aaaggttaga gcattctctt aggtgtagac ctagtgaaga gacagatggc 1860 tcctggttta actcctcaag tggctctgat gcagttttag taaaggctgt cttaagactt 1920 actgattcat tagccaggga agcacagcca atccatggag gtgacccaga agatattata 1980 cacaagttgt taagatctgc ttgggccgag tgggttaggg aaaaggcaga cgctgccgat 2040 agcgtgtgca atggtagttc tgcagtagaa caagagggat caagaatggt ccatgataaa 2100 cagacctgtc tattattggc tagaatgatc gaaatttctg ccggtagggc agctggtgaa 2160 gcagccagtg aggacggcga tagaagaata attcaattaa caggctccat ctgcgacagt 2220 cttaagcaaa aaatgctagt ttcacaggac cctgaaaaaa atgaagagat gatgtctcac 2280 gtggatgacg aattgaagtt gaggattaga gagttcgttc aatatttgct tagactaggt 2340 gaaaaaaaga ctggatctag cgaaaccagg caaacatttt taagtatagt gaaatcatgt 2400 tactatgctg ctcattgccc acctcatgtc gttgatagac acattagtag agtgattttc 2460 gagccagtaa gtgccgcaaa gtaaccgcgg 2490
SEQ ID NO:40
Z mays
MVLSSSCTTV PHLSSLAWQ LGPWSSRIKK KTDTVAVPAA AGRWRRALAR AQHTSESAAV 60
AKGSSLTPIV RTDAESRRTR WPTDDDDAEP LVDEIRAMLT SMSDGDISVS AYDTAWVGLV 120
PRLDGGEGPQ FPAAVRWIRN NQLPDGSWGD AALFSAYDRL INTLACVVTL TRWSLEPEMR 180
GRGLSFLGRN MWKLATEDEE SMPIGFELAF PSLIELAKSL GVHDFPYDHQ ALQGIYSSRE 240
IKMKRIPKEV MHTVPTSILH SLEGMPGLDW AKLLKLQSSD GSFLFSPAAT AYALMNTGDD 300
RCFSYIDRTV KKFNGGVPNV YPVDLFEHIW AVDRLERLGI SRYFQKEIEQ CMDYVNRHWT 360
EDGICWARNS DVKEVDDTAM AFRLLRLHGY SVSPDVFKNF EKDGEFFAFV GQSNQAVTGM 420
YNLNRASQIS FPGEDVLHRA GAFSYEFLRR KEAEGALRDK WIISKDLPGE WYTLDFPWY 480
GNLPRVEARD YLEQYGGGDD VWIGKTLYRM PLVNNDVYLE LARMDFNHCQ ALHQLEWQGL 540
KRWYTENRLM DFGVAQEDAL RAYFLAAASV YEPCRAAERL AWARAAILAN AVSTHLRNSP 600
SFRERLEHSL RCRPSEETDG SWFNSSSGSD AVLVKAVLRL TDSLAREAQP IHGGDPEDII 660
HKLLRSAWAE WVREKADAAD SVCNGSSAVE QEGSRMVHDK QTCLLLARMI EISAGRAAGE 720
AASEDGDRRI IQLTGSICDS LKQKMLVSQD PEKNEEMMSH VDDELKLRIR EFVQYLLRLG 780 EKKTGSSETR QTFLSIVKSC YYAAHCPPHV VDRHISRVIF EPVSAAK 827
SEQ ID NO:41
cttcttcact aaatacttag acagagaaaa cagagctttt taaagccatg tctcttcagt 60 atcatgttct aaactccatt ccaagtacaa cctttctcag ttctactaaa acaacaatat 120 cttcttcttt ccttaccatc tcaggatctc ctctcaatgt cgctagagac aaatccagaa 180 gcggttccat acattgttca aagcttcgaa ctcaagaata cattaattct caagaggttc 240 aacatgattt gcctctaata catgagtggc aacagcttca aggagaagat gctcctcaga 300 ttagtgttgg aagtaatagt aatgcattca aagaagcagt gaagagtgtg aaaacgatct 360 tgagaaacct aacggacggg gaaattacga tatcggctta cgatacagct tgggttgcat 420 tgatcgatgc cggagataaa actccggcgt ttccctccgc cgtgaaatgg atcgccgaga 480 accaactttc cgatggttct tggggagatg cgtatctctt ctcttatcat gatcgtctca 540 tcaataccct tgcatgcgtc gttgctctaa gatcatggaa tctctttcct catcaatgca 600 acaaaggaat cacgtttttc cgggaaaata ttgggaagct agaagacgaa aatgatgagc 660 atatgccaat cggattcgaa gtagcattcc catcgttgct tgagatagct cgaggaataa 720 acattgatgt accgtacgat tctccggtct taaaagatat atacgccaag aaagagctaa 780 agcttacaag gataccaaaa gagataatgc acaagatacc aacaacattg ttgcatagtt 840 tggaggggat gcgtgattta gattgggaaa agctcttgaa acttcaatct caagacggat 900 ctttcctctt ctctccttcc tctaccgctt ttgcattcat gcagacccga gacagtaact 960 gcctcgagta tttgcgaaat gccgtcaaac gtttcaatgg aggagttccc aatgtctttc 1020 ccgtggatct tttcgagcac atatggatag tggatcggtt acaacgttta gggatatcga 1080 gatactttga agaagagatt aaagagtgtc ttgactatgt ccacagatat tggaccgaca 1140 atggcatatg ttgggctaga tgttcccatg tccaagacat cgatgataca gccatggcat 1200 ttaggctctt aagacaacat ggataccaag tgtccgcaga tgtattcaag aactttgaga 1260 aagagggaga gtttttctgc tttgtggggc aatcaaacca agcagtaacc ggtatgttca 1320 acctataccg ggcatcacaa ttggcgtttc caagggaaga gatattgaaa aacgccaaag 1380 agttttctta taattatctg ctagaaaaac gggagagaga ggagttgatt gataagtgga 1440 ttataatgaa agacttacct ggcgagattg ggtttgcgtt agagattcca tggtacgcaa 1500 gcttgcctcg agtagagacg agattctata ttgatcaata tggtggagaa aacgacgttt 1560 ggattggcaa gactctttat aggatgccat acgtgaacaa taatggatat ctggaattag 1620 caaaacaaga ttacaacaat tgccaagctc agcatcagct cgaatgggac atattccaaa 1680 agtggtatga agaaaatagg ttaagtgagt ggggtgtgcg cagaagtgag cttctcgagt 1740 gttactactt agcggctgca actatatttg aatcagaaag gtcacatgag agaatggttt 1800 gggctaagtc aagtgtattg gttaaagcca tttcttcttc ttttggggaa tcctctgact 1860 ccagaagaag cttctccgat cagtttcatg aatacattgc caatgctcga cgaagtgatc 1920 atcactttaa tgacaggaac atgagattgg accgaccagg atcggttcag gccagtcggc 1980 ttgccggagt gttaatcggg actttgaatc aaatgtcttt tgaccttttc atgtctcatg 2040 gccgtgacgt taacaatctc ctctatctat cgtggggaga ttggatggaa aaatggaaac 2100 tatatggaga tgaaggagaa ggagagctca tggtgaagat gataattcta atgaagaaca 2160 atgacctaac taacttcttc acccacactc acttcgttcg tctcgcggaa atcatcaatc 2220 gaatctgtct tcctcgccaa tacttaaagg caaggagaaa cgatgagaag gagaagacaa 2280 taaagagtat ggagaaggag atggggaaaa tggttgagtt agcattgtcg gagagtgaca 2340 catttcgtga cgtcagcatc acgtttcttg atgtagcaaa agcattttac tactttgctt 2400 tatgtggcga tcatctccaa actcacatct ccaaagtctt gtttcaaaaa gtctagtaac 2460 ctcatcatca tcatcgatcc attaacaatc agtggatcga tgtatccata gatgcgtgaa 2520 taatatttca tgtagagaag gagaacaaat tagatcatgt agggttatca 2570
SEQ ID NO:42
Arabidopsis thaliana
MSLQYHVLNS IPSTTFLSST KTTISSSFLT ISGSPLNVAR DKSRSGSIHC SKLRTQEYIN 60
SQEVQHDLPL IHEWQQLQGE DAPQISVGSN SNAFKEAVKS VKTILRNLTD GEITISAYDT 120
AWVALIDAGD KTPAFPSAVK WIAENQLSDG SWGDAYLFSY HDRLINTLAC WALRSWNLF 180
PHQCNKGITF FRENIGKLED ENDEHMPIGF EVAFPSLLEI ARGINIDVPY DSPVLKDIYA 240
KKELKLTRIP KEIMHKIPTT LLHSLEGMRD LDWEKLLKLQ SQDGSFLFSP SSTAFAFMQT 300
RDSNCLEYLR NAVKRFNGGV PNVFPVDLFE HIWIVDRLQR LGISRYFEEE IKECLDYVHR 360 YWTDNGICWA RCSHVQDIDD TAMAFRLLRQ HGYQVSADVF KNFEKEGEFF CFVGQSNQAV 420
TGMFNLYRAS QLAFPREEIL KNAKEFSYNY LLEKREREEL IDKWIIMKDL PGEIGFALEI 480
PWYASLPRVE TRFYIDQYGG ENDVWIGKTL YRMPYVNNNG YLELAKQDYN NCQAQHQLEW 540
DIFQKWYEEN RLSEWGVRRS ELLECYYLAA ATIFESERSH ERMVWAKSSV LVKAISSSFG 600
ESSDSRRSFS DQFHEYIANA RRSDHHFNDR NMRLDRPGSV QASRLAGVLI GTLNQMSFDL 660
FMSHGRDVNN LLYLSWGDWM EKWKLYGDEG EGELMVKMII LMKNNDLTNF FTHTHFVRLA 720
EIINRICLPR QYLKARRNDE KEKTIKSMEK EMGKMVELAL SESDTFRDVS ITFLDVAKAF 780
YYFALCGDHL QTHISKVLFQ KV 802
SEQ ID NO:43
atgaatttga gtttgtgtat agcatctcca ctattgacca aatctaatag accagctgct 60 ttatcagcaa ttcatacagc tagtacatcc catggtggcc aaaccaaccc tacgaatctg 120 ataatcgata cgaccaagga gagaatacaa aaacaattca aaaatgttga aatttcagtt 180 tcttcttatg atactgcgtg ggttgccatg gttccatcac ctaattctcc aaagtctcca 240 tgtttcccag aatgtttgaa ttggctgatt aacaaccagt tgaatgatgg atcttggggt 300 ttagtcaatc acacgcacaa tcacaaccat ccacttttga aagattcttt atcctcaact 360 ttggcttgca tcgtggccct aaagagatgg aacgtaggtg aggatcagat taacaagggg 420 cttagtttca ttgaatctaa cttggcttcc gcgactgaaa aatctcaacc atctccaata 480 ggattcgata tcatctttcc aggtctgtta gagtacgcca aaaatctaga tatcaactta 540 ctgtctaagc aaactgattt ctcactaatg ttacacaaga gagaattaga acaaaagaga 600 tgtcattcaa acgaaatgga tggttaccta gcttatatct ctgaaggtct tggtaatctt 660 tacgattgga atatggtgaa aaagtaccag atgaaaaatg gctcagtttt caattcccct 720 tctgcaactg cggcagcatt cattaaccat caaaatccag gatgcctgaa ctatttgaat 780 tcactactag acaaattcgg caacgcagtt ccaactgtat accctcacga tttgtttatc 840 agattgagta tggtggatac aattgaaaga cttggtatat cccaccactt tagagtcgag 900 atcaaaaatg ttttggatga gacataccgt tgttgggtgg agagagatga acaaatcttt 960 atggatgttg tgacgtgcgc gttggccttt agattgttgc gtattaacgg ttacgaagtt 1020 agtccagatc cacttgccga aattacaaac gaattagctt taaaggatga atacgccgct 1080 cttgaaacat atcatgcgtc acatatcctt taccaagagg acttatcatc tggaaaacaa 1140 attcttaaat ctgctgattt cctgaaggaa atcatatcca ctgatagtaa tagactgtcc 1200 aaactgatcc ataaagaggt tgaaaatgca cttaagttcc ctattaacac cggcttagaa 1260 cgtattaaca caagacgtaa catccagctt tacaacgtag acaatactag aatcttgaaa 1320 accacttacc attcttccaa catatcaaac actgattacc taagattagc tgttgaagat 1380 ttctacacat gtcagtctat ctatagagaa gagctgaaag gattagagag atgggtcgtt 1440 gagaataagc tagatcaatt gaaatttgcc agacaaaaga cagcttattg ttacttctca 1500 gttgccgcca ctttatcaag tccagaattg tcagatgcac gtatttcttg ggctaaaaac 1560 ggaattttga caactgttgt tgatgatttc tttgatattg gcgggacaat cgacgaattg 1620 acaaacctga ttcaatgcgt tgaaaagtgg aatgtcgatg tcgataaaga ctgttgctca 1680 gaacatgtta gaatactgtt cttggctctg aaagatgcta tctgttggat cggggatgag 1740 gctttcaaat ggcaagctag agatgtgacg tctcacgtca ttcaaacctg gctagaactg 1800 atgaactcta tgttgagaga agcaatttgg actagagatg catacgttcc tacattaaac 1860 gagtatatgg aaaacgctta tgtctccttt gctttgggtc ctatcgttaa gcctgccata 1920 tactttgtag gaccaaagct atccgaggaa atcgtcgaat catcagaata ccataacttg 1980 ttcaagttaa tgtccacaca aggcagatta cttaatgata ttcattcttt caaaagagag 2040 tttaaggaag gaaagttaaa tgctgttgct ctgcatcttt ctaatggcga aagtggtaaa 2100 gtcgaagagg aagtagttga ggaaatgatg atgatgatca aaaacaagag aaaggagttg 2160 atgaaactaa tcttcgaaga gaacggttca attgttccta gagcatgtaa ggatgcattt 2220 tggaacatgt gtcatgtgct aaactttttc tacgcaaacg acgatggttt tactgggaac 2280 acaatactag atacagtaaa agacatcata tacaaccctt tggtcttagt aaacgaaaac 2340 gaggagcaaa gataa 2355
SEQ ID NO:44
Stevia rebaudiana
MNLSLCIASP LLTKSNRPAA LSAIHTASTS HGGQTNPTNL IIDTTKERIQ KQFKNVEISV 60
SSYDTAWVAM VPSPNSPKSP CFPECLNWLI NNQLNDGSWG LVNHTHNHNH PLLKDSLSST 120 LACIVALKRW NVGEDQINKG LSFIESNLAS ATEKSQPSPI GFDIIFPGLL EYAKNLDINL 180
LSKQTDFSLM LHKRELEQKR CHSNEMDGYL AYISEGLGNL YDWNMVKKYQ MKNGSVFNSP 240
SATAAAFINH QNPGCLNYLN SLLDKFGNAV PTVYPHDLFI RLSMVDTIER LGISHHFRVE 300
IKNVLDETYR CWVERDEQIF MDWTCALAF RLLRINGYEV SPDPLAEITN ELALKDEYAA 360
LETYHASHIL YQEDLSSGKQ ILKSADFLKE IISTDSNRLS KLIHKEVENA LKFPINTGLE 420
RINTRRNIQL YNVDNTRILK TTYHSSNISN TDYLRLAVED FYTCQSIYRE ELKGLERWW 480
ENKLDQLKFA RQKTAYCYFS VAATLSSPEL SDARISWAKN GILTTWDDF FDIGGTIDEL 540
TNLIQCVEKW NVDVDKDCCS EHVRILFLAL KDAICWIGDE AFKWQARDVT SHVIQTWLEL 600
MNSMLREAIW TRDAYVPTLN EYMENAYVSF ALGPIVKPAI YFVGPKLSEE IVESSEYHNL 660
FKLMSTQGRL LNDIHSFKRE FKEGKLNAVA LHLSNGESGK VEEEWEEMM MMIKNKRKEL 720
MKLIFEENGS IVPRACKDAF WNMCHVLNFF YANDDGFTGN TILDTVKDII YNPLVLVNEN 780
EEQR 784
SEQ ID NO:45
atgaatctgt ccctttgtat agctagtcca ctgttgacaa aatcttctag accaactgct 60 ctttctgcaa ttcatactgc cagtactagt catggaggtc aaacaaaccc aacaaatttg 120 ataatcgata ctactaagga gagaatccaa aagctattca aaaatgttga aatctcagta 180 tcatcttatg acaccgcatg ggttgcaatg gtgccatcac ctaattcccc aaaaagtcca 240 tgttttccag agtgcttgaa ttggttaatc aataatcagt taaacgatgg ttcttggggt 300 ttagtcaacc acactcataa ccacaatcat ccattattga aggactcttt atcatcaaca 360 ttagcctgta ttgttgcatt gaaaagatgg aatgtaggtg aagatcaaat caacaagggt 420 ttatcattca tagaatccaa tctagcttct gctaccgaca aatcacaacc atctccaatc 480 gggttcgaca taatcttccc tggtttgctg gagtatgcca aaaaccttga tatcaactta 540 ctgtctaaac aaacagattt ctctttgatg ctacacaaaa gagagttaga gcagaaaaga 600 tgccattcta acgaaattga cgggtactta gcatatatct cagaaggttt gggtaatttg 660 tatgactgga acatggtcaa aaagtatcag atgaaaaatg gatccgtatt caattctcct 720 tctgcaactg ccgcagcatt cattaatcat caaaaccctg ggtgtcttaa ctacttgaac 780 tcactattag ataagtttgg aaatgcagtt ccaacagtct atcctttgga cttgtacatc 840 agattatcta tggttgacac tatagagaga ttaggtattt ctcatcattt cagagttgag 900 atcaaaaatg ttttggacga gacatacaga tgttgggtcg aaagagatga gcaaatcttt 960 atggatgtcg tgacctgcgc tctggctttt agattgctaa ggatacacgg atacaaagta 1020 tctcctgatc aactggctga gattacaaac gaactggctt tcaaagacga atacgccgca 1080 ttagaaacat accatgcatc ccaaatactt taccaggaag acctaagttc aggaaaacaa 1140 atcttgaagt ctgcagattt cctgaaaggc attctgtcta cagatagtaa taggttgtct 1200 aaattgatac acaaggaagt agaaaacgca ctaaagtttc ctattaacac tggtttagag 1260 agaatcaata ctaggagaaa cattcagctg tacaacgtag ataatacaag gattcttaag 1320 accacctacc atagttcaaa catttccaac acctattact taagattagc tgtcgaagac 1380 ttttacactt gtcaatcaat ctacagagag gagttaaagg gcctagaaag atgggtagtt 1440 caaaacaagt tggatcaact gaagtttgct agacagaaga cagcatactg ttatttctct 1500 gttgctgcta ccctttcatc cccagaattg tctgatgcca gaataagttg ggccaaaaat 1560 ggtattctta caactgtagt cgatgatttc tttgatattg gaggtactat tgatgaactg 1620 acaaatctta ttcaatgtgt tgaaaagtgg aacgtggatg tagataagga ttgctgcagt 1680 gaacatgtga gaatactttt cctggctcta aaagatgcaa tatgttggat tggcgacgag 1740 gccttcaagt ggcaagctag agatgttaca tctcatgtca tccaaacttg gcttgaactg 1800 atgaactcaa tgctaagaga agcaatctgg acaagagatg catacgttcc aacattgaac 1860 gaatacatgg aaaacgctta cgtctcattt gccttgggtc ctattgttaa gccagccata 1920 tactttgttg ggccaaagtt atccgaagag attgttgagt cttccgaata tcataaccta 1980 ttcaagttaa tgtcaacaca aggcagactt ctgaacgata tccactcctt caaaagagaa 2040 ttcaaggaag gtaagctaaa cgctgttgct ttgcacttgt ctaatggtga atctggcaaa 2100 gtggaagagg aagtcgttga ggaaatgatg atgatgatca aaaacaagag aaaggaattg 2160 atgaaattga ttttcgagga aaatggttca atcgtaccta gagcttgtaa agatgctttt 2220 tggaatatgt gccatgttct taacttcttt tacgctaatg atgatggctt cactggaaat 2280 acaatattgg atacagttaa agatatcatc tacaacccac ttgttttggt caatgagaac 2340 gaggaacaaa gataa 2355 SEQ ID NO:46
Stevia rebaudiana
MNLSLCIASP LLTKSSRPTA LSAIHTASTS HGGQTNPTNL IIDTTKERIQ KLFKNVEISV 60
SSYDTAWVAM VPSPNSPKSP CFPECLNWLI NNQLNDGSWG LVNHTHNHNH PLLKDSLSST 120
LACIVALKRW NVGEDQINKG LSFIESNLAS ATDKSQPSPI GFDIIFPGLL EYAKNLDINL 180
LSKQTDFSLM LHKRELEQKR CHSNEIDGYL AYISEGLGNL YDWNMVKKYQ MKNGSVFNSP 240
SATAAAFINH QNPGCLNYLN SLLDKFGNAV PTVYPLDLYI RLSMVDTIER LGISHHFRVE 300
IKNVLDETYR CWVERDEQIF MDWTCALAF RLLRIHGYKV SPDQLAEITN ELAFKDEYAA 360
LETYHASQIL YQEDLSSGKQ ILKSADFLKG ILSTDSNRLS KLIHKEVENA LKFPINTGLE 420
RINTRRNIQL YNVDNTRILK TTYHSSNISN TYYLRLAVED FYTCQSIYRE ELKGLERWW 480
QNKLDQLKFA RQKTAYCYFS VAATLSSPEL SDARISWAKN GILTTWDDF FDIGGTIDEL 540
TNLIQCVEKW NVDVDKDCCS EHVRILFLAL KDAICWIGDE AFKWQARDVT SHVIQTWLEL 600
MNSMLREAIW TRDAYVPTLN EYMENAYVSF ALGPIVKPAI YFVGPKLSEE IVESSEYHNL 660
FKLMSTQGRL LNDIHSFKRE FKEGKLNAVA LHLSNGESGK VEEEWEEMM MMIKNKRKEL 720
MKLIFEENGS IVPRACKDAF WNMCHVLNFF YANDDGFTGN TILDTVKDII YNPLVLVNEN 780
EEQR 784
SEQ ID NO:47
atggctatgc cagtgaagct aacacctgcg tcattatcct taaaagctgt gtgctgcaga 60 ttctcatccg gtggccatgc tttgagattc gggagtagtc tgccatgttg gagaaggacc 120 cctacccaaa gatctacttc ttcctctact actagaccag ctgccgaagt gtcatcaggt 180 aagagtaaac aacatgatca ggaagctagt gaagcgacta tcagacaaca attacaactt 240 gtggatgtcc tggagaatat gggaatatcc agacattttg ctgcagagat aaagtgcata 300 ctagacagaa cttacagatc ttggttacaa agacacgagg aaatcatgct ggacactatg 360 acatgtgcta tggcttttag aatcctaaga ttgaacggat acaacgtttc atcagatgaa 420 ctataccacg ttgtagaggc atctggtctg cataattctt tgggtgggta tcttaacgat 480 accagaacac tacttgaatt acacaaggct tcaacagtta gtatctctga ggatgaatct 540 atcttagatt caattggctc tagatccaga acattgctta gagaacaatt ggagtctggt 600 ggcgcactga gaaagccttc tttattcaaa gaggttgaac atgcactgga tggacctttt 660 tacaccacac ttgatagact tcatcatagg tggaatattg aaaacttcaa cattattgag 720 caacacatgt tggagactcc atacttatct aaccagcata catcaaggga tatcctagca 780 ttgtcaatta gagatttttc ctcctcacaa ttcacttatc aacaagagct acagcatctg 840 gagagttggg ttaaggaatg tagattagat caactacagt tcgcaagaca gaaattagcg 900 tacttttacc tatcagccgc aggcaccatg ttttctcctg agctttctga tgcgagaaca 960 ttatgggcca aaaacggggt gttgacaact attgttgatg atttctttga tgttgccggt 1020 tctaaagagg aattggaaaa cttagtcatg ctggtcgaaa tgtgggatga acatcacaaa 1080 gttgaattct attctgagca ggtcgaaatc atcttctctt ccatctacga ttctgtcaac 1140 caattgggtg agaaggcctc tttggttcaa gacagatcaa ttacaaaaca ccttgttgaa 1200 atatggttag acttgttaaa gtccatgatg acggaagttg aatggagact gtcaaaatac 1260 gtgcctacag aaaaggaata catgattaat gcctctctta tcttcggcct aggtccaatc 1320 gttttaccag ctttgtattt cgttggtcca aagatttcag aaagtatagt aaaggaccca 1380 gaatatgatg aattgttcaa actaatgtca acatgtggta gattgttgaa tgacgtgcaa 1440 acgttcgaaa gagaatacaa tgagggtaaa ctgaattctg tcagtctatt ggttcttcac 1500 ggaggcccaa tgtctatttc agacgcaaag aggaaattac aaaagcctat tgatacgtgt 1560 agaagagatc ttctttcttt ggtccttaga gaagagtctg tagtaccaag accatgtaag 1620 gaactattct ggaaaatgtg taaagtgtgc tatttctttt actcaacaac tgatgggttt 1680 tctagtcaag tcgaaagagc aaaagaggta gacgctgtca taaatgagcc actgaagttg 1740 caaggttctc atacactggt atctgatgtt taa 1773
SEQ ID NO:48
Zea mays
MAMPVKLTPA SLSLKAVCCR FSSGGHALRF GSSLPCWRRT PTQRSTSSST TRPAAEVSSG 60
KSKQHDQEAS EATIRQQLQL VDVLENMGIS RHFAAEIKCI LDRTYRSWLQ RHEEIMLDTM 120
TCAMAFRILR LNGYNVSSDE LYHVVEASGL HNSLGGYLND TRTLLELHKA STVSISEDES 180
ILDSIGSRSR TLLREQLESG GALRKPSLFK EVEHALDGPF YTTLDRLHHR WNIENFNI IE 240 QHMLETPYLS NQHTSRDILA LSIRDFSSSQ FTYQQELQHL ESWVKECRLD QLQFARQKLA 300
YFYLSAAGTM FSPELSDART LWAKNGVLTT IVDDFFDVAG SKEELENLVM LVEMWDEHHK 360
VEFYSEQVEI IFSSIYDSVN QLGEKASLVQ DRSITKHLVE IWLDLLKSMM TEVEWRLSKY 420
VPTEKEYMIN ASLIFGLGPI VLPALYFVGP KISESIVKDP EYDELFKLMS TCGRLLNDVQ 480
TFEREYNEGK LNSVSLLVLH GGPMSISDAK RKLQKPIDTC RRDLLSLVLR EESWPRPCK 540
ELFWKMCKVC YFFYSTTDGF SSQVERAKEV DAVINEPLKL QGSHTLVSDV 590
SEQ ID NO:49
atgcagaact tccatggtac aaaggaaagg atcaaaaaga tgtttgacaa gattgaattg 60 tccgtttctt cttatgatac agcctgggtt gcaatggtcc catcccctga ttgcccagaa 120 acaccttgtt ttccagaatg tactaaatgg atcctagaaa atcagttggg tgatggtagt 180 tggtcacttc ctcatggcaa tccacttcta gttaaagatg cattatcttc cactcttgct 240 tgtattctgg ctcttaaaag atggggaatc ggtgaggaac agattaacaa aggactgaga 300 ttcatagaac tcaactctgc tagtgtaacc gataacgaac aacacaaacc aattggattt 360 gacattatct ttccaggtat gattgaatac gctatagact tagacctgaa tctaccacta 420 aaaccaactg acattaactc catgttgcat cgtagagccc ttgaattgac atcaggtgga 480 ggcaaaaatc tagaaggtag aagagcttac ttggcctacg tctctgaagg aatcggtaag 540 ctgcaagatt gggaaatggc tatgaaatac caacgtaaaa acggatctct gttcaatagt 600 ccatcaacaa ctgcagctgc attcatccat atacaagatg ctgaatgcct ccactatatt 660 cgttctcttc tccagaaatt tggaaacgca gtccctacaa tataccctct cgatatctat 720 gccagacttt caatggtaga tgccctggaa cgtcttggta ttgatagaca tttcagaaag 780 gagagaaagt tcgttctgga tgaaacatac agattttggt tgcaaggaga agaggagatt 840 ttctccgata acgcaacctg tgctttggcc ttcagaatat tgagacttaa tggttacgat 900 gtctctcttg aagatcactt ctctaactct ctgggcggtt acttaaagga ctcaggagca 960 gctttagaac tgtacagagc cctccaattg tcttacccag acgagtccct cctggaaaag 1020 caaaattcta gaacttctta cttcttaaaa caaggtttat ccaatgtctc cctctgtggt 1080 gacagattgc gtaaaaacat aattggagag gtgcatgatg ctttaaactt ttccgaccac 1140 gctaacttac aaagattagc tattcgtaga aggattaagc attacgctac tgacgataca 1200 aggattctaa aaacttccta cagatgctca acaatcggta accaagattt tctaaaactt 1260 gcagtggaag atttcaatat ctgtcaatca atacaaagag aggaattcaa gcatattgaa 1320 agatgggtcg ttgaaagacg tctagacaag ttaaagttcg ctagacaaaa agaggcctat 1380 tgctatttct cagccgcagc aacattgttt gcccctgaat tgtctgatgc tagaatgtct 1440 tgggccaaaa atggtgtatt gacaactgtg gttgatgatt tcttcgatgt cggaggctct 1500 gaagaggaat tagttaactt gatagaattg atcgagcgtt gggatgtgaa tggcagtgca 1560 gatttttgta gtgaggaagt tgagattatc tattctgcta tccactcaac tatctctgaa 1620 ataggtgata agtcatttgg ctggcaaggt agagatgtaa agtctcaagt tatcaagatc 1680 tggctggact tattgaaatc aatgttaact gaagctcaat ggtcttcaaa caagtctgtt 1740 cctaccctag atgagtatat gacaaccgcc catgtttcat tcgcacttgg tccaattgta 1800 cttccagcct tatacttcgt tggcccaaag ttgtcagaag aggttgcagg tcatcctgaa 1860 ctactaaacc tctacaaagt cacatctact tgtggcagac tactgaatga ttggagaagt 1920 tttaagagag aatccgagga aggtaagctc aacgctatta gtttatacat gatccactcc 1980 ggtggtgctt ctacagaaga ggaaacaatc gaacatttca aaggtttgat tgattctcag 2040 agaaggcaac tgttacaatt ggtgttgcaa gagaaggata gtatcatacc tagaccatgt 2100 aaagatctat tttggaatat gattaagtta ttacacactt tctacatgaa agatgatggc 2160 ttcacctcaa atgagatgag gaatgtagtt aaggcaatca ttaacgaacc aatctcactg 2220 gatgaattat ga 2232
SEQ ID NO:50
Populus trichocarpa
MSCIRPWFCP SSISATLTDP ASKLVTGEFK TTSLNFHGTK ERIKKMFDKI ELSVSSYDTA 60
WVAMVPSPDC PETPCFPECT KWILENQLGD GSWSLPHGNP LLVKDALSST LACILALKRW 120
GIGEEQINKG LRFIELNSAS VTDNEQHKPI GFDIIFPGMI EYAKDLDLNL PLKPTDINSM 180
LHRRALELTS GGGKNLEGRR AYLAYVSEGI GKLQDWEMAM KYQRKNGSLF NSPSTTAAAF 240
IHIQDAECLH YIRSLLQKFG NAVPTIYPLD IYARLSMVDA LERLGIDRHF RKERKFVLDE 300
TYRFWLQGEE EIFSDNATCA LAFRILRLNG YDVSLEDHFS NSLGGYLKDS GAALELYRAL 360 QLSYPDESLL EKQNSRTSYF LKQGLSNVSL CGDRLRKNII GEVHDALNFP DHANLQRLAI 420
RRRIKHYATD DTRILKTSYR CSTIGNQDFL KLAVEDFNIC QSIQREEFKH IERWVVERRL 480
DKLKFARQKE AYCYFSAAAT LFAPELSDAR MSWAKNGVLT TWDDFFDVG GSEEELVNLI 540
ELIERWDVNG SADFCSEEVE IIYSAIHSTI SEIGDKSFGW QGRDVKSHVI KIWLDLLKSM 600
LTEAQWSSNK SVPTLDEYMT TAHVSFALGP IVLPALYFVG PKLSEEVAGH PELLNLYKVM 660
STCGRLLNDW RSFKRESEEG KLNAISLYMI HSGGASTEEE TIEHFKGLID SQRRQLLQLV 720
LQEKDSIIPR PCKDLFWNMI KLLHTFYMKD DGFTSNEMRN WKAIINEPI SLDEL 775
SEQ ID NO:51
A. thaliana
atgtctatca accttcgctc ctccggttgt tcgtctccga tctcagctac tttggaacga 60 ggattggact cagaagtaca gacaagagct aacaatgtga gctttgagca aacaaaggag 120 aagattagga agatgttgga gaaagtggag ctttctgttt cggcctacga tactagttgg 180 gtagcaatgg ttccatcacc gagctcccaa aatgctccac ttttcccaca gtgtgtgaaa 240 tggttattgg ataatcaaca tgaagatgga tcttggggac ttgataacca tgaccatcaa 300 tctcttaaga aggatgtgtt atcatctaca ctggctagta tcctcgcgtt aaagaagtgg 360 ggaattggtg aaagacaaat aaacaagggt ctccagttta ttgagctgaa ttctgcatta 420 gtcactgatg aaaccataca gaaaccaaca gggtttgata ttatatttcc tgggatgatt 480 aaatatgcta gagatttgaa tctgacgatt ccattgggct cagaagtggt ggatgacatg 540 atacgaaaaa gagatctgga tcttaaatgt gatagtgaaa agttttcaaa gggaagagaa 600 gcatatctgg cctatgtttt agaggggaca agaaacctaa aagattggga tttgatagtc 660 aaatatcaaa ggaaaaatgg gtcactgttt gattctccag ccacaacagc agctgctttt 720 actcagtttg ggaatgatgg ttgtctccgt tatctctgtt ctctccttca gaaattcgag 780 gctgcagttc cttcagttta tccatttgat caatatgcac gccttagtat aattgtcact 840 cttgaaagct taggaattga tagagatttc aaaaccgaaa tcaaaagcat attggatgaa 900 acctatagat attggcttcg tggggatgaa gaaatatgtt tggacttggc cacttgtgct 960 ttggctttcc gattattgct tgctcatggc tatgatgtgt cttacgatcc gctaaaacca 1020 tttgcagaag aatctggttt ctctgatact ttggaaggat atgttaagaa tacgttttct 1080 gtgttagaat tatttaaggc tgctcaaagt tatccacatg aatcagcttt gaagaagcag 1140 tgttgttgga ctaaacaata tctggagatg gaattgtcca gctgggttaa gacctctgtt 1200 cgagataaat acctcaagaa agaggtcgag gatgctcttg cttttccctc ctatgcaagc 1260 ctagaaagat cagatcacag gagaaaaata ctcaatggtt ctgctgtgga aaacaccaga 1320 gttacaaaaa cctcatatcg tttgcacaat atttgcacct ctgatatcct gaagttagct 1380 gtggatgact tcaatttctg ccagtccata caccgtgaag aaatggaacg tcttgatagg 1440 tggattgtgg agaatagatt gcaggaactg aaatttgcca gacagaagct ggcttactgt 1500 tatttctctg gggctgcaac tttattttct ccagaactat ctgatgctcg tatatcgtgg 1560 gccaaaggtg gagtacttac aacggttgta gacgacttct ttgatgttgg agggtccaaa 1620 gaagaactgg aaaacctcat acacttggtc gaaaagtggg atttgaacgg tgttcctgag 1680 tacagctcag aacatgttga gatcatattc tcagttctaa gggacaccat tctcgaaaca 1740 ggagacaaag cattcaccta tcaaggacgc aatgtgacac accacattgt gaaaatttgg 1800 ttggatctgc tcaagtctat gttgagagaa gccgagtggt ccagtgacaa gtcaacacca 1860 agcttggagg attacatgga aaatgcgtac atatcatttg cattaggacc aattgtcctc 1920 ccagctacct atctgatcgg acctccactt ccagagaaga cagtcgatag ccaccaatat 1980 aatcagctct acaagctcgt gagcactatg ggtcgtcttc taaatgacat acaaggtttt 2040 aagagagaaa gcgcggaagg gaagctgaat gcggtttcat tgcacatgaa acacgagaga 2100 gacaatcgca gcaaagaagt gatcatagaa tcgatgaaag gtttagcaga gagaaagagg 2160 gaagaattgc ataagctagt tttggaggag aaaggaagtg tggttccaag ggaatgcaaa 2220 gaagcgttct tgaaaatgag caaagtgttg aacttatttt acaggaagga cgatggattc 2280 acatcaaatg atctgatgag tcttgttaaa tcagtgatct acgagcctgt tagcttacag 2340 aaagaatctt taacttga 2358
SEQ ID NO:52
A. thaliana
MSINLRSSGC SSPISATLER GLDSEVQTRA NNVSFEQTKE KIRKMLEKVE LSVSAYDTSW 60
VAMVPSPSSQ NAPLFPQCVK WLLDNQHEDG SWGLDNHDHQ SLKKDVLSST LASILALKKW 120 GIGERQINKG LQFIELNSAL VTDETIQKPT GFDIIFPGMI KYARDLNLTI PLGSEWDDM 180
IRKRDLDLKC DSEKFSKGRE AYLAYVLEGT RNLKDWDLIV KYQRKNGSLF DSPATTAAAF 240
TQFGNDGCLR YLCSLLQKFE AAVPSVYPFD QYARLSIIVT LESLGIDRDF KTEIKSILDE 300
TYRYWLRGDE EICLDLATCA LAFRLLLAHG YDVSYDPLKP FAEESGFSDT LEGYVKNTFS 360
VLELFKAAQS YPHESALKKQ CCWTKQYLEM ELSSWVKTSV RDKYLKKEVE DALAFPSYAS 420
LERSDHRRKI LNGSAVENTR VTKTSYRLHN ICTSDILKLA VDDFNFCQSI HREEMERLDR 480
WIVENRLQEL KFARQKLAYC YFSGAATLFS PELSDARISW AKGGVLTTW DDFFDVGGSK 540
EELENLIHLV EKWDLNGVPE YSSEHVEIIF SVLRDTILET GDKAFTYQGR NVTHHIVKIW 600
LDLLKSMLRE AEWSSDKSTP SLEDYMENAY ISFALGPIVL PATYLIGPPL PEKTVDSHQY 660
NQLYKLVSTM GRLLNDIQGF KRESAEGKLN AVSLHMKHER DNRSKEVIIE SMKGLAERKR 720
EELHKLVLEE KGSWPRECK EAFLKMSKVL NLFYRKDDGF TSNDLMSLVK SVIYEPVSLQ 780
KESLT 785
SEQ ID NO:53
atggaatttg atgaaccatt ggttgacgaa gcaagatctt tagtgcagcg tactttacaa 60 gattatgatg acagatacgg cttcggtact atgtcatgtg ctgcttatga tacagcctgg 120 gtgtctttag ttacaaaaac agtcgatggg agaaaacaat ggcttttccc agagtgtttt 180 gaatttctac tagaaacaca atctgatgcc ggaggatggg aaatcgggaa ttcagcacca 240 atcgacggta tattgaatac agctgcatcc ttacttgctc taaaacgtca cgttcaaact 300 gagcaaatca tccaacctca acatgaccat aaggatctag caggtagagc tgaacgtgcc 360 gctgcatctt tgagagcaca attggctgca ttggatgtgt ctacaactga acacgtcggt 420 tttgagataa ttgttcctgc aatgctagac ccattagaag ccgaagatcc atctctagtt 480 ttcgattttc cagctaggaa acctttgatg aagattcatg atgctaagat gagtagattc 540 aggccagaat acttgtatgg caaacaacca atgaccgcct tacattcatt agaggctttc 600 ataggcaaaa tcgacttcga taaggtaaga caccaccgta cccatgggtc tatgatgggt 660 tctccttcat ctaccgcagc ctacttaatg cacgcttcac aatgggatgg tgactcagag 720 gcttacctta gacacgtgat taaacacgca gcagggcagg gaactggtgc tgtaccatct 780 gctttcccat caacacattt tgagtcatct tggattctta ccacattgtt tagagctgga 840 ttttcagctt ctcatcttgc ctgtgatgag ttgaacaagt tggtcgagat acttgagggc 900 tcattcgaga aggaaggtgg ggcaatcggt tacgctccag ggtttcaagc agatgttgat 960 gatactgcta aaacaataag tacattagca gtccttggaa gagatgctac accaagacaa 1020 atgatcaagg tatttgaagc taatacacat tttagaacat accctggtga aagagatcct 1080 tctttgacag ctaattgtaa tgctctatca gccttactac accaaccaga tgcagcaatg 1140 tatggatctc aaattcaaaa gattaccaaa tttgtctgtg actattggtg gaagtctgat 1200 ggtaagatta aagataagtg gaacacttgc tacttgtacc catctgtctt attagttgag 1260 gttttggttg atcttgttag tttattggag cagggtaaat tgcctgatgt tttggatcaa 1320 gagcttcaat acagagtcgc catcacattg ttccaagcat gtttaaggcc attactagac 1380 caagatgccg aaggatcatg gaacaagtct atcgaagcca cagcctacgg catccttatc 1440 ctaactgaag ctaggagagt ttgtttcttc gacagattgt ctgagccatt gaatgaggca 1500 atccgtagag gtatcgcttt cgccgactct atgtctggaa ctgaagctca gttgaactac 1560 atttggatcg aaaaggttag ttacgcacct gcattattga ctaaatccta tttgttagca 1620 gcaagatggg ctgctaagtc tcctttaggc gcttccgtag gctcttcttt gtggactcca 1680 ccaagagaag gattggataa gcatgtcaga ttattccatc aagctgagtt attcagatcc 1740 cttccagaat gggaattaag agcctccatg attgaagcag ctttgttcac accacttcta 1800 agagcacata gactagacgt tttccctaga caagatgtag gtgaagacaa atatcttgat 1860 gtagttccat tcttttggac tgccgctaac aacagagata gaacttacgc ttccactcta 1920 ttcctttacg atatgtgttt tatcgcaatg ttaaacttcc agttagacga attcatggag 1980 gccacagccg gtatcttatt cagagatcat atggatgatt tgaggcaatt gattcatgat 2040 cttttggcag agaaaacttc cccaaagagt tctggtagaa gtagtcaggg cacaaaagat 2100 gctgactcag gtatagagga agacgtgtca atgtccgatt cagcttcaga ttcccaggat 2160 agaagtccag aatacgactt ggttttcagt gcattgagta cctttacaaa acatgtcttg 2220 caacacccat ctatacaaag tgcctctgta tgggatagaa aactacttgc tagagagatg 2280 aaggcttact tacttgctca tatccaacaa gcagaagatt caactccatt gtctgaattg 2340 aaagatgtgc ctcaaaagac tgatgtaaca agagtttcta catctactac taccttcttt 2400 aactgggtta gaacaacttc cgcagaccat atatcctgcc catactcctt ccactttgta 2460 gcatgccatc taggcgcagc attgtcacct aaagggtcta acggtgattg ctatccttca 2520 gctggtgaga agttcttggc agctgcagtc tgcagacatt tggccaccat gtgtagaatg 2580 tacaacgatc ttggatcagc tgaacgtgat tctgatgaag gtaatttgaa ctccttggac 2640 ttccctgaat tcgccgattc cgcaggaaac ggagggatag aaattcagaa ggccgctcta 2700 ttaaggttag ctgagtttga gagagattca tacttagagg ccttccgtcg tttacaagat 2760 gaatccaata gagttcacgg tccagccggt ggtgatgaag ccagattgtc cagaaggaga 2820 atggcaatcc ttgaattctt cgcccagcag gtagatttgt acggtcaagt atacgtcatt 2880 agggatattt ccgctcgtat tcctaaaaac gaggttgaga aaaagagaaa attggatgat 2940 gctttcaatt ga 2952
SEQ ID NO:54
Phomopsis amygdali
MEFDEPLVDE ARSLVQRTLQ DYDDRYGFGT MSCAAYDTAW VSLVTKTVDG RKQWLFPECF 60
EFLLETQSDA GGWEIGNSAP IDGILNTAAS LLALKRHVQT EQIIQPQHDH KDLAGRAERA 120
AASLRAQLAA LDVSTTEHVG FEIIVPAMLD PLEAEDPSLV FDFPARKPLM KIHDAKMSRF 180
RPEYLYGKQP MTALHSLEAF IGKIDFDKVR HHRTHGSMMG SPSSTAAYLM HASQWDGDSE 240
AYLRHVIKHA AGQGTGAVPS AFPSTHFESS WILTTLFRAG FSASHLACDE LNKLVEILEG 300
SFEKEGGAIG YAPGFQADVD DTAKTISTLA VLGRDATPRQ MIKVFEANTH FRTYPGERDP 360
SLTANCNALS ALLHQPDAAM YGSQIQKITK FVCDYWWKSD GKIKDKWNTC YLYPSVLLVE 420
VLVDLVSLLE QGKLPDVLDQ ELQYRVAITL FQACLRPLLD QDAEGSWNKS IEATAYGILI 480
LTEARRVCFF DRLSEPLNEA IRRGIAFADS MSGTEAQLNY IWIEKVSYAP ALLTKSYLLA 540
ARWAAKSPLG ASVGSSLWTP PREGLDKHVR LFHQAELFRS LPEWELRASM IEAALFTPLL 600
RAHRLDVFPR QDVGEDKYLD VVPFFWTAAN NRDRTYASTL FLYDMCFIAM LNFQLDEFME 660
ATAGILFRDH MDDLRQLIHD LLAEKTSPKS SGRSSQGTKD ADSGIEEDVS MSDSASDSQD 720
RSPEYDLVFS ALSTFTKHVL QHPSIQSASV WDRKLLAREM KAYLLAHIQQ AEDSTPLSEL 780
KDVPQKTDVT RVSTSTTTFF NWVRTTSADH ISCPYSFHFV ACHLGAALSP KGSNGDCYPS 840
AGEKFLAAAV CRHLATMCRM YNDLGSAERD SDEGNLNSLD FPEFADSAGN GGIEIQKAAL 900
LRLAEFERDS YLEAFRRLQD ESNRVHGPAG GDEARLSRRR MAILEFFAQQ VDLYGQVYVI 960
RDISARIPKN EVEKKRKLDD AFN 983
SEQ ID NO:55
atggcttcta gtacacttat ccaaaacaga tcatgtggcg tcacatcatc tatgtcaagt 60 tttcaaatct tcagaggtca accactaaga tttcctggca ctagaacccc agctgcagtt 120 caatgcttga aaaagaggag atgccttagg ccaaccgaat ccgtactaga atcatctcct 180 ggctctggtt catatagaat agtaactggc ccttctggaa ttaaccctag ttctaacggg 240 cacttgcaag agggttcctt gactcacagg ttaccaatac caatggaaaa atctatcgat 300 aacttccaat ctactctata tgtgtcagat atttggtctg aaacactaca gagaactgaa 360 tgtttgctac aagtaactga aaacgtccag atgaatgagt ggattgagga aattagaatg 420 tactttagaa atatgacttt aggtgaaatt tccatgtccc cttacgacac tgcttgggtg 480 gctagagttc cagcgttgga cggttctcat gggcctcaat tccacagatc tttgcaatgg 540 attatcgaca accaattacc agatggggac tggggcgaac cttctctttt cttgggttac 600 gatagagttt gtaatacttt agcctgtgtg attgcgttga aaacatgggg tgttggggca 660 caaaacgttg aaagaggaat tcagttccta caatctaaca tatacaagat ggaggaagat 720 gacgctaatc atatgccaat aggattcgaa atcgtattcc ctgctatgat ggaagatgcc 780 aaagcattag gtttggattt gccatacgat gctactattt tgcaacagat ttcagccgaa 840 agagagaaaa agatgaaaaa gatcccaatg gcaatggtgt acaaataccc aaccacttta 900 cttcactcct tagaaggctt gcatagagaa gttgattgga ataagttgtt acaattacaa 960 tctgaaaatg gtagttttct ttattcacct gcttcaaccg catgcgcctt aatgtacact 1020 aaggacgtta aatgttttga ttacttaaac cagttgttga tcaagttcga ccacgcatgc 1080 ccaaatgtat atccagtcga tctattcgaa agattatgga tggttgacag attgcagaga 1140 ttagggatct ccagatactt tgaaagagag attagagatt gtttacaata cgtctacaga 1200 tattggaaag attgtggaat cggatgggct tctaactctt ccgtacaaga tgttgatgat 1260 acagccatgg cgtttagact tttaaggact catggtttcg acgtaaagga agattgcttt 1320 agacagtttt tcaaggacgg agaattcttc tgcttcgcag gccaatcatc tcaagcagtt 1380 acaggcatgt ttaatctttc aagagccagt caaacattgt ttccaggaga atctttattg 1440 aaaaaggcta gaaccttctc tagaaacttc ttgagaacaa agcatgagaa caacgaatgt 1500 ttcgataaat ggatcattac taaagatttg gctggtgaag tcgagtataa cttgaccttc 1560 ccatggtatg cctctttgcc tagattagaa cataggacat acttagatca atatggaatc 1620 gatgatatct ggataggcaa atctttatac aaaatgcctg ctgttaccaa cgaagttttc 1680 ctaaagttgg caaaggcaga ctttaacatg tgtcaagctc tacacaaaaa ggaattggaa 1740 caagtgataa agtggaacgc gtcctgtcaa ttcagagatc ttgaattcgc cagacaaaaa 1800 tcagtagaat gctattttgc tggtgcagcc acaatgttcg aaccagaaat ggttcaagct 1860 agattagtct gggcaagatg ttgtgtattg acaactgtct tagacgatta ctttgaccac 1920 gggacacctg ttgaggaact tagagtgttt gttcaagctg tcagaacatg gaatccagag 1980 ttgatcaacg gtttgccaga gcaagctaaa atcttgttta tgggcttata caaaacagtt 2040 aacacaattg cagaggaagc attcatggca cagaaaagag acgtccatca tcatttgaaa 2100 cactattggg acaagttgat aacaagtgcc ctaaaggagg ccgaatgggc agagtcaggt 2160 tacgtcccaa catttgatga atacatggaa gtagctgaaa tttctgttgc tctagaacca 2220 attgtctgta gtaccttgtt ctttgcgggt catagactag atgaggatgt tctagatagt 2280 tacgattacc atctagttat gcatttggta aacagagtcg gtagaatctt gaatgatata 2340 caaggcatga agagggaggc ttcacaaggt aagatctcat cagttcaaat ctacatggag 2400 gaacatccat ctgttccatc tgaggccatg gcgatcgctc atcttcaaga gttagttgat 2460 aattcaatgc agcaattgac atacgaagtt cttaggttca ctgcggttcc aaaaagttgt 2520 aagagaatcc acttgaatat ggctaaaatc atgcatgcct tctacaagga tactgatgga 2580 ttctcatccc ttactgcaat gacaggattc gtcaaaaagg ttcttttcga acctgtgcct 2640 gagtaa 2646
SEQ ID NO:56
Physcomitrella patens
MASSTLIQNR SCGVTSSMSS FQIFRGQPLR FPGTRTPAAV QCLKKRRCLR PTESVLESSP 60
GSGSYRIVTG PSGINPSSNG HLQEGSLTHR LPIPMEKSID NFQSTLYVSD IWSETLQRTE 120
CLLQVTENVQ MNEWIEEIRM YFRNMTLGEI SMSPYDTAWV ARVPALDGSH GPQFHRSLQW 180
IIDNQLPDGD WGEPSLFLGY DRVCNTLACV IALKTWGVGA QNVERGIQFL QSNIYKMEED 240
DANHMPIGFE IVFPAMMEDA KALGLDLPYD ATILQQISAE REKKMKKIPM AMVYKYPTTL 300
LHSLEGLHRE VDWNKLLQLQ SENGSFLYSP ASTACALMYT KDVKCFDYLN QLLIKFDHAC 360
PNVYPVDLFE RLWMVDRLQR LGISRYFERE IRDCLQYVYR YWKDCGIGWA SNSSVQDVDD 420
TAMAFRLLRT HGFDVKEDCF RQFFKDGEFF CFAGQSSQAV TGMFNLSRAS QTLFPGESLL 480
KKARTFSRNF LRTKHENNEC FDKWIITKDL AGEVEYNLTF PWYASLPRLE HRTYLDQYGI 540
DDIWIGKSLY KMPAVTNEVF LKLAKADFNM CQALHKKELE QVIKWNASCQ FRDLEFARQK 600
SVECYFAGAA TMFEPEMVQA RLVWARCCVL TTVLDDYFDH GTPVEELRVF VQAVRTWNPE 660
LINGLPEQAK ILFMGLYKTV NTIAEEAFMA QKRDVHHHLK HYWDKLITSA LKEAEWAESG 720
YVPTFDEYME VAEISVALEP IVCSTLFFAG HRLDEDVLDS YDYHLVMHLV NRVGRILNDI 780
QGMKREASQG KISSVQIYME EHPSVPSEAM AIAHLQELVD NSMQQLTYEV LRFTAVPKSC 840
KRIHLNMAKI MHAFYKDTDG FSSLTAMTGF VKKVLFEPVP E 881
SEQ ID NO:57
atgcctggta aaattgaaaa tggtacccca aaggacctca agactggaaa tgattttgtt 60 tctgctgcta agagtttact agatcgagct ttcaaaagtc atcattccta ctacggatta 120 tgctcaactt catgtcaagt ttatgataca gcttgggttg caatgattcc aaaaacaaga 180 gataatgtaa aacagtggtt gtttccagaa tgtttccatt acctcttaaa aacacaagcc 240 gcagatggct catggggttc attgcctaca acacagacag cgggtatcct agatacagcc 300 tcagctgtgc tggcattatt gtgccacgca caagagcctt tacaaatatt ggatgtatct 360 ccagatgaaa tggggttgag aatagaacac ggtgtcacat ccttgaaacg tcaattagca 420 gtttggaatg atgtggagga caccaaccat attggcgtcg agtttatcat accagcctta 480 ctttccatgc tagaaaagga attagatgtt ccatcttttg aatttccatg taggtccatc 540 ttagagagaa tgcacgggga gaaattaggt catttcgacc tggaacaagt ttacggcaag 600 ccaagctcat tgttgcactc attggaagca tttctcggta agctagattt tgatcgacta 660 tcacatcacc tataccacgg cagtatgatg gcatctccat cttcaacggc tgcttatctt 720 attggggcta caaaatggga tgacgaagcc gaagattacc taagacatgt aatgcgtaat 780 ggtgcaggac atgggaatgg aggtatttct ggtacatttc caactactca tttcgaatgt 840 agctggatta tagcaacgtt gttaaaggtt ggctttactt tgaagcaaat tgacggcgat 900 ggcttaagag gtttatcaac catcttactt gaggcgcttc gtgatgagaa tggtgtcata 960 ggctttgccc ctagaacagc agatgtagat gacacagcca aagctctatt ggccttgtca 1020 ttggtaaacc agccagtgtc acctgatatc atgattaagg tctttgaggg caaagaccat 1080 tttaccactt ttggttcaga aagagatcca tcattgactt ccaacctgca cgtcctttta 1140 tctttactta aacaatctaa cttgtctcaa taccatcctc aaatcctcaa aacaacatta 1200 ttcacttgta gatggtggtg gggttccgat cattgtgtca aagacaaatg gaatttgagt 1260 cacctatatc caactatgtt gttggttgaa gccttcactg aagtgctcca tctcattgac 1320 ggtggtgaat tgtctagtct gtttgatgaa tcctttaagt gtaagattgg tcttagcatc 1380 tttcaagcgg tacttagaat aatcctcacc caagacaacg acggctcttg gagaggatac 1440 agagaacaga cgtgttacgc aatattggct ttagttcaag cgagacatgt atgctttttc 1500 actcacatgg ttgacagact gcaatcatgt gttgatcgag gtttctcatg gttgaaatct 1560 tgctcttttc attctcaaga cctgacttgg acctctaaaa cagcttatga agtgggtttc 1620 gtagctgaag catataaact agctgcttta caatctgctt ccctggaggt tcctgctgcc 1680 accattggac attctgtcac gtctgccgtt ccatcaagtg atcttgaaaa atacatgaga 1740 ttggtgagaa aaactgcgtt attctctcca ctggatgagt ggggtctaat ggcttctatc 1800 atcgaatctt catttttcgt accattactg caggcacaaa gagttgaaat ataccctaga 1860 gataatatca aggtggacga agataagtac ttgtctatta tcccattcac atgggtcgga 1920 tgcaataata ggtctagaac tttcgcaagt aacagatggc tatacgatat gatgtacctt 1980 tcattactcg gctatcaaac cgacgagtac atggaagctg tagctgggcc agtgtttggg 2040 gatgtttcct tgttacatca aacaattgat aaggtgattg ataatacaat gggtaacctt 2100 gcgagagcca atggaacagt acacagtggt aatggacatc agcacgaatc tcctaatata 2160 ggtcaagtcg aggacacctt gactcgtttc acaaattcag tcttgaatca caaagacgtc 2220 cttaactcta gctcatctga tcaagatact ttgagaagag agtttagaac attcatgcac 2280 gctcatataa cacaaatcga agataactca cgattcagta agcaagcctc atccgatgcg 2340 ttttcctctc ctgaacaatc ttactttcaa tgggtgaact caactggtgg ctcacatgtc 2400 gcttgcgcct attcatttgc cttctctaat tgcctcatgt ctgcaaattt gttgcagggt 2460 aaagacgcat ttccaagcgg aacgcaaaag tacttaatct cctctgttat gagacatgcc 2520 acaaacatgt gtagaatgta taacgacttt ggctctattg ccagagacaa cgctgagaga 2580 aatgttaata gtattcattt tcctgagttt actctctgta acggaacttc tcaaaaccta 2640 gatgaaagga aggaaagact tctgaaaatc gcaacttacg aacaagggta tttggataga 2700 gcactagagg ccttggaaag acagagtaga gatgatgccg gagacagagc tggatctaaa 2760 gatatgagaa agttgaaaat cgttaagtta ttctgtgatg ttacggactt atacgatcag 2820 ctctacgtta tcaaagattt gtcatcctct atgaagtaa 2859
SEQ ID NO:58
Gibberella fujikuroi
MPGKIENGTP KDLKTGNDFV SAAKSLLDRA FKSHHSYYGL CSTSCQVYDT AWVAMIPKTR 60
DNVKQWLFPE CFHYLLKTQA ADGSWGSLPT TQTAGILDTA SAVLALLCHA QEPLQILDVS 120
PDEMGLRIEH GVTSLKRQLA VWNDVEDTNH IGVEFIIPAL LSMLEKELDV PSFEFPCRSI 180
LERMHGEKLG HFDLEQVYGK PSSLLHSLEA FLGKLDFDRL SHHLYHGSMM ASPSSTAAYL 240
IGATKWDDEA EDYLRHVMRN GAGHGNGGIS GTFPTTHFEC SWIIATLLKV GFTLKQIDGD 300
GLRGLSTILL EALRDENGVI GFAPRTADVD DTAKALLALS LVNQPVSPDI MIKVFEGKDH 360
FTTFGSERDP SLTSNLHVLL SLLKQSNLSQ YHPQILKTTL FTCRWWWGSD HCVKDKWNLS 420
HLYPTMLLVE AFTEVLHLID GGELSSLFDE SFKCKIGLSI FQAVLRI ILT QDNDGSWRGY 480
REQTCYAILA LVQARHVCFF THMVDRLQSC VDRGFSWLKS CSFHSQDLTW TSKTAYEVGF 540
VAEAYKLAAL QSASLEVPAA TIGHSVTSAV PSSDLEKYMR LVRKTALFSP LDEWGLMASI 600
IESSFFVPLL QAQRVEIYPR DNIKVDEDKY LSIIPFTWVG CNNRSRTFAS NRWLYDMMYL 660
SLLGYQTDEY MEAVAGPVFG DVSLLHQTID KVIDNTMGNL ARANGTVHSG NGHQHESPNI 720
GQVEDTLTRF TNSVLNHKDV LNSSSSDQDT LRREFRTFMH AHITQIEDNS RFSKQASSDA 780
FSSPEQSYFQ WVNSTGGSHV ACAYSFAFSN CLMSANLLQG KDAFPSGTQK YLISSVMRHA 840
TNMCRMYNDF GSIARDNAER NVNSIHFPEF TLCNGTSQNL DERKERLLKI ATYEQGYLDR 900
ALEALERQSR DDAGDRAGSK DMRKLKIVKL FCDVTDLYDQ LYVIKDLSSS MK 952
SEQ ID NO:59 S. rebaudiana
atggatgctg tgacgggttt gttaactgtc ccagcaaccg ctataactat tggtggaact 60 gctgtagcat tggcggtagc gctaatcttt tggtacctga aatcctacac atcagctaga 120 agatcccaat caaatcatct tccaagagtg cctgaagtcc caggtgttcc attgttagga 180 aatctgttac aattgaagga gaaaaagcca tacatgactt ttacgagatg ggcagcgaca 240 tatggaccta tctatagtat caaaactggg gctacaagta tggttgtggt atcatctaat 300 gagatagcca aggaggcatt ggtgaccaga ttccaatcca tatctacaag gaacttatct 360 aaagccctga aagtacttac agcagataag acaatggtcg caatgtcaga ttatgatgat 420 tatcataaaa cagttaagag acacatactg accgccgtct tgggtcctaa tgcacagaaa 480 aagcatagaa ttcacagaga tatcatgatg gataacatat ctactcaact tcatgaattc 540 gtgaaaaaca acccagaaca ggaagaggta gaccttagaa aaatctttca atctgagtta 600 ttcggcttag ctatgagaca agccttagga aaggatgttg aaagtttgta cgttgaagac 660 ctgaaaatca ctatgaatag agacgaaatc tttcaagtcc ttgttgttga tccaatgatg 720 ggagcaatcg atgttgattg gagagacttc tttccatacc taaagtgggt cccaaacaaa 780 aagttcgaaa atactattca acaaatgtac atcagaagag aagctgttat gaaatcttta 840 atcaaagagc acaaaaagag aatagcgtca ggcgaaaagc taaatagtta tatcgattac 900 cttttatctg aagctcaaac tttaaccgat cagcaactat tgatgtcctt gtgggaacca 960 atcattgaat cttcagatac aacaatggtc acaacagaat gggcaatgta cgaattagct 1020 aaaaacccta aattgcaaga taggttgtac agagacatta agtccgtctg tggatctgaa 1080 aagataaccg aagagcatct atcacagctg ccttacatta cagctatttt ccacgaaaca 1140 ctgagaagac actcaccagt tcctatcatt cctctaagac atgtacatga agataccgtt 1200 ctaggcggct accatgttcc tgctggcaca gaacttgccg ttaacatcta cggttgcaac 1260 atggacaaaa acgtttggga aaatccagag gaatggaacc cagaaagatt catgaaagag 1320 aatgagacaa ttgattttca aaagacgatg gccttcggtg gtggtaagag agtttgtgct 1380 ggttccttgc aagccctttt aactgcatct attgggattg ggagaatggt tcaagagttc 1440 gaatggaaac tgaaggatat gactcaagag gaagtgaaca cgataggcct aactacacaa 1500 atgttaagac cattgagagc tattatcaaa cctaggatct aa 1542
SEQ ID NO:60
S. rebaudiana
MDAVTGLLTV PATAITIGGT AVALAVALIF WYLKSYTSAR RSQSNHLPRV PEVPGVPLLG 60
NLLQLKEKKP YMTFTRWAAT YGPIYSIKTG ATSMVWSSN EIAKEALVTR FQSISTRNLS 120
KALKVLTADK TMVAMSDYDD YHKTVKRHIL TAVLGPNAQK KHRIHRDIMM DNISTQLHEF 180
VKNNPEQEEV DLRKIFQSEL FGLAMRQALG KDVESLYVED LKITMNRDEI FQVLVVDPMM 240
GAIDVDWRDF FPYLKWVPNK KFENTIQQMY IRREAVMKSL IKEHKKRIAS GEKLNSYIDY 300
LLSEAQTLTD QQLLMSLWEP IIESSDTTMV TTEWAMYELA KNPKLQDRLY RDIKSVCGSE 360
KITEEHLSQL PYITAI FHET LRRHSPVPII PLRHVHEDTV LGGYHVPAGT ELAVNIYGCN 420
MDKNVWENPE EWNPERFMKE NETIDFQKTM AFGGGKRVCA GSLQALLTAS IGIGRMVQEF 480
EWKLKDMTQE EVNTIGLTTQ MLRPLRAIIK PRI 513
SEQ ID NO:61
aagcttacta gtaaaatgga cggtgtcatc gatatgcaaa ccattccatt gagaaccgct 60 attgctattg gtggtactgc tgttgctttg gttgttgcat tatacttttg gttcttgaga 120 tcctacgctt ccccatctca tcattctaat catttgccac cagtacctga agttccaggt 180 gttccagttt tgggtaattt gttgcaattg aaagaaaaaa agccttacat gaccttcacc 240 aagtgggctg aaatgtatgg tccaatctac tctattagaa ctggtgctac ttccatggtt 300 gttgtctctt ctaacgaaat cgccaaagaa gttgttgtta ccagattccc atctatctct 360 accagaaaat tgtcttacgc cttgaaggtt ttgaccgaag ataagtctat ggttgccatg 420 tctgattatc acgattacca taagaccgtc aagagacata ttttgactgc tgttttgggt 480 ccaaacgccc aaaaaaagtt tagagcacat agagacacca tgatggaaaa cgtttccaat 540 gaattgcatg ccttcttcga aaagaaccca aatcaagaag tcaacttgag aaagatcttc 600 caatcccaat tattcggttt ggctatgaag caagccttgg gtaaagatgt tgaatccatc 660 tacgttaagg atttggaaac caccatgaag agagaagaaa tcttcgaagt tttggttgtc 720 gatccaatga tgggtgctat tgaagttgat tggagagact ttttcccata cttgaaatgg 780 gttccaaaca agtccttcga aaacatcatc catagaatgt acactagaag agaagctgtt 840 atgaaggcct tgatccaaga acacaagaaa agaattgcct ccggtgaaaa cttgaactcc 900 tacattgatt acttgttgtc tgaagcccaa accttgaccg ataagcaatt attgatgtct 960 ttgtgggaac ctattatcga atcttctgat accactatgg ttactactga atgggctatg 1020 tacgaattgg ctaagaatcc aaacatgcaa gacagattat acgaagaaat ccaatccgtt 1080 tgcggttccg aaaagattac tgaagaaaac ttgtcccaat tgccatactt gtacgctgtt 1140 ttccaagaaa ctttgagaaa gcactgtcca gttcctatta tgccattgag atatgttcac 1200 gaaaacaccg ttttgggtgg ttatcatgtt ccagctggta ctgaagttgc tattaacatc 1260 tacggttgca acatggataa gaaggtctgg gaaaatccag aagaatggaa tccagaaaga 1320 ttcttgtccg aaaaagaatc catggacttg tacaaaacta tggcttttgg tggtggtaaa 1380 agagtttgcg ctggttcttt acaagccatg gttatttctt gcattggtat cggtagattg 1440 gtccaagatt ttgaatggaa gttgaaggat gatgccgaag aagatgttaa cactttgggt 1500 ttgactaccc aaaagttgca tccattattg gccttgatta acccaagaaa gtaactcgag 1560 ccgcgg 1566
SEQ ID NO:62
Lactuca sativa
MDGVIDMQTI PLRTAIAIGG TAVALWALY FWFLRSYASP SHHSNHLPPV PEVPGVPVLG 60 NLLQLKEKKP YMTFTKWAEM YGPIYSIRTG ATSMVWSSN EIAKEWVTR FPSISTRKLS 120 YALKVLTEDK SMVAMSDYHD YHKTVKRHIL TAVLGPNAQK KFRAHRDTMM ENVSNELHAF 180 FEKNPNQEVN LRKIFQSQLF GLAMKQALGK DVESIYVKDL ETTMKREEIF EVLWDPMMG 240 AIEVDWRDFF PYLKWVPNKS FENI IHRMYT RREAVMKALI QEHKKRIASG ENLNSYIDYL 300 LSEAQTLTDK QLLMSLWEPI IESSDTTMVT TEWAMYELAK NPNMQDRLYE EIQSVCGSEK 360 ITEENLSQLP YLYAVFQETL RKHCPVPIMP LRYVHENTVL GGYHVPAGTE VAINIYGCNM 420 DKKVWENPEE WNPERFLSEK ESMDLYKTMA FGGGKRVCAG SLQAMVISCI GIGRLVQDFE 480 WKLKDDAEED VNTLGLTTQK LHPLLALINP RK 512
SEQ ID NO:63
R. suavissimus
atggccaccc tccttgagca tttccaagct atgccctttg ccatccctat tgcactggct 60 gctctgtctt ggctgttcct cttttacatc aaagtttcat tcttttccaa caagagtgct 120 caggctaagc tccctcctgt gccagtggtt cctgggctgc cggtgattgg gaatttactg 180 caactcaagg agaagaaacc ctaccagact tttacaaggt gggctgagga gtatggacca 240 atctattcta tcaggactgg tgcttccacc atggtcgttc tcaataccac ccaagttgca 300 aaagaggcca tggtgaccag atatttatcc atctcaacca gaaagctatc aaacgcacta 360 aagattctta ctgctgataa atgtatggtt gcaataagtg actacaacga ttttcacaag 420 atgataaagc gatacatact ctcaaatgtt cttggaccta gtgctcagaa gcgtcaccgg 480 agcaacagag ataccttgag agctaatgtc tgcagccgat tgcattctca agtaaagaac 540 tctcctcgag aagctgtgaa tttcagaaga gtttttgagt gggaactctt tggaattgca 600 ttgaagcaag cctttggaaa ggacatagaa aagcccattt atgtggagga acttggcact 660 acactgtcaa gagatgagat ctttaaggtt ctagtgcttg acataatgga gggtgcaatt 720 gaggttgatt ggagagattt cttcccttac ctgagatgga ttccgaatac gcgcatggaa 780 acaaaaattc agcgactcta tttccgcagg aaagcagtga tgactgccct gatcaacgag 840 cagaagaagc gaattgcttc aggagaggaa atcaactgtt atatcgactt cttgcttaag 900 gaagggaaga cactgacaat ggaccaaata agtatgttgc tttgggagac ggttattgaa 960 acagcagata ctacaatggt aacgacagaa tgggctatgt atgaagttgc taaagactca 1020 aagcgtcagg atcgtctcta tcaggaaatc caaaaggttt gtggatcgga gatggttaca 1080 gaggaatact tgtcccaact gccgtacctg aatgcagttt tccatgaaac gctaaggaag 1140 cacagtccgg ctgcgttagt tcctttaaga tatgcacatg aagataccca actaggaggt 1200 tactacattc cagctggaac tgagattgct ataaacatat acgggtgtaa catggacaag 1260 catcaatggg aaagccctga ggaatggaaa ccggagagat ttttggaccc gaaatttgat 1320 cctatggatt tgtacaagac catggctttt ggggctggaa agagggtatg tgctggttct 1380 cttcaggcaa tgttaatagc gtgcccgacg attggtaggc tggtgcagga gtttgagtgg 1440 aagctgagag atggagaaga agaaaatgta gatactgttg ggctcaccac tcacaaacgc 1500 tatccaatgc atgcaatcct gaagccaaga agtta 1535 SEQ ID NO:64
R. suavissimus
atggctacct tgttggaaca ttttcaagct atgccattcg ctattccaat tgctttggct 60 gctttgtctt ggttgttttt gttctacatc aaggtttctt tcttctccaa caaatccgct 120 caagctaaat tgccaccagt tccagttgtt ccaggtttgc cagttattgg taatttgttg 180 caattgaaag aaaagaagcc ataccaaacc ttcactagat gggctgaaga atatggtcca 240 atctactcta ttagaactgg tgcttctact atggttgtct tgaacactac tcaagttgcc 300 aaagaagcta tggttaccag atacttgtct atctctacca gaaagttgtc caacgccttg 360 aaaattttga ccgctgataa gtgcatggtt gccatttctg attacaacga tttccacaag 420 atgatcaaga gatatatctt gtctaacgtt ttgggtccat ctgcccaaaa aagacataga 480 tctaacagag ataccttgag agccaacgtt tgttctagat tgcattccca agttaagaac 540 tctccaagag aagctgtcaa ctttagaaga gttttcgaat gggaattatt cggtatcgct 600 ttgaaacaag ccttcggtaa ggatattgaa aagccaatct acgtcgaaga attgggtact 660 actttgtcca gagatgaaat cttcaaggtt ttggtcttgg acattatgga aggtgccatt 720 gaagttgatt ggagagattt tttcccatac ttgcgttgga ttccaaacac cagaatggaa 780 actaagatcc aaagattata ctttagaaga aaggccgtta tgaccgcctt gattaacgaa 840 caaaagaaaa gaattgcctc cggtgaagaa atcaactgct acatcgattt cttgttgaaa 900 gaaggtaaga ccttgaccat ggaccaaatc tctatgttgt tgtgggaaac cgttattgaa 960 actgctgata ccacaatggt tactactgaa tgggctatgt acgaagttgc taaggattct 1020 aaaagacaag acagattata ccaagaaatc caaaaggtct gcggttctga aatggttaca 1080 gaagaatact tgtcccaatt gccatacttg aatgctgttt tccacgaaac tttgagaaaa 1140 cattctccag ctgctttggt tccattgaga tatgctcatg aagatactca attgggtggt 1200 tattacattc cagccggtac tgaaattgcc attaacatct acggttgcaa catggacaaa 1260 caccaatggg aatctccaga agaatggaag ccagaaagat ttttggatcc taagtttgac 1320 ccaatggact tgtacaaaac tatggctttt ggtgctggta aaagagtttg cgctggttct 1380 ttacaagcta tgttgattgc ttgtccaacc atcggtagat tggttcaaga atttgaatgg 1440 aagttgagag atggtgaaga agaaaacgtt gatactgttg gtttgaccac ccataagaga 1500 tatccaatgc atgctatttt gaagccaaga tcttaa 1536
SEQ ID NO:65
aagcttacta gtaaaatggc ctccatcacc catttcttac aagattttca agctactcca 60 ttcgctactg cttttgctgt tggtggtgtt tctttgttga tattcttctt cttcatccgt 120 ggtttccact ctactaagaa aaacgaatat tacaagttgc caccagttcc agttgttcca 180 ggtttgccag ttgttggtaa tttgttgcaa ttgaaagaaa agaagccata caagactttc 240 ttgagatggg ctgaaattca tggtccaatc tactctatta gaactggtgc ttctaccatg 300 gttgttgtta actctactca tgttgccaaa gaagctatgg ttaccagatt ctcttcaatc 360 tctaccagaa agttgtccaa ggctttggaa ttattgacct ccaacaaatc tatggttgcc 420 acctctgatt acaacgaatt tcacaagatg gtcaagaagt acatcttggc cgaattattg 480 ggtgctaatg ctcaaaagag acacagaatt catagagaca ccttgatcga aaacgtcttg 540 aacaaattgc atgcccatac caagaattct ccattgcaag ctgttaactt cagaaagatc 600 ttcgaatctg aattattcgg tttggctatg aagcaagcct tgggttatga tgttgattcc 660 ttgttcgttg aagaattggg tactaccttg tccagagaag aaatctacaa cgttttggtc 720 agtgacatgt tgaagggtgc tattgaagtt gattggagag actttttccc atacttgaaa 780 tggatcccaa acaagtcctt cgaaatgaag attcaaagat tggcctctag aagacaagcc 840 gttatgaact ctattgtcaa agaacaaaag aagtccattg cctctggtaa gggtgaaaac 900 tgttacttga attacttgtt gtccgaagct aagactttga ccgaaaagca aatttccatt 960 ttggcctggg aaaccattat tgaaactgct gatacaactg ttgttaccac tgaatgggct 1020 atgtacgaat tggctaaaaa cccaaagcaa caagacagat tatacaacga aatccaaaac 1080 gtctgcggta ctgataagat taccgaagaa catttgtcca agttgcctta cttgtctgct 1140 gtttttcacg aaaccttgag aaagtattct ccatctccat tggttccatt gagatacgct 1200 catgaagata ctcaattggg tggttattat gttccagccg gtactgaaat tgctgttaat 1260 atctacggtt gcaacatgga caagaatcaa tgggaaactc cagaagaatg gaagccagaa 1320 agatttttgg acgaaaagta cgatccaatg gacatgtaca agactatgtc ttttggttcc 1380 ggtaaaagag tttgcgctgg ttctttacaa gctagtttga ttgcttgtac ctccatcggt 1440 agattggttc aagaatttga atggagattg aaagacggtg aagttgaaaa cgttgatacc 1500 ttgggtttga ctacccataa gttgtatcca atgcaagcta tcttgcaacc tagaaactga 1560 ctcgagccgc gg 1572
SEQ ID NO:66
Castanea mollissima
MASITHFLQD FQATPFATAF AVGGVSLLIF FFFIRGFHST KKNEYYKLPP VPWPGLPW 60
GNLLQLKEKK PYKTFLRWAE IHGPIYSIRT GASTMWVNS THVAKEAMVT RFSSISTRKL 120
SKALELLTSN KSMVATSDYN EFHKMVKKYI LAELLGANAQ KRHRIHRDTL IENVLNKLHA 180
HTKNSPLQAV NFRKIFESEL FGLAMKQALG YDVDSLFVEE LGTTLSREEI YNVLVSDMLK 240
GAIEVDWRDF FPYLKWIPNK SFEMKIQRLA SRRQAVMNSI VKEQKKSIAS GKGENCYLNY 300
LLSEAKTLTE KQISILAWET IIETADTTW TTEWAMYELA KNPKQQDRLY NEIQNVCGTD 360
KITEEHLSKL PYLSAVFHET LRKYSPSPLV PLRYAHEDTQ LGGYYVPAGT EIAVNIYGCN 420
MDKNQWETPE EWKPERFLDE KYDPMDMYKT MSFGSGKRVC AGSLQASLIA CTSIGRLVQE 480
FEWRLKDGEV ENVDTLGLTT HKLYPMQAIL QPRN 514
SEQ ID NO:67
atgatttcct tgttgttggg ttttgttgtc tcctccttct tgtttatctt cttcttgaaa 60 aaattgttgt tcttcttcag tcgtcacaaa atgtccgaag tttctagatt gccatctgtt 120 ccagttccag gttttccatt gattggtaac ttgttgcaat tgaaagaaaa gaagccacac 180 aagactttca ccaagtggtc tgaattatat ggtccaatct actctatcaa gatgggttcc 240 tcttctttga tcgtcttgaa ctctattgaa accgccaaag aagctatggt cagtagattc 300 tcttcaatct ctaccagaaa gttgtctaac gctttgactg ttttgacctg caacaaatct 360 atggttgcta cctctgatta cgatgacttt cataagttcg tcaagagatg cttgttgaac 420 ggtttgttgg gtgctaatgc tcaagaaaga aaaagacatt acagagatgc cttgatcgaa 480 aacgttacct ctaaattgca tgcccatacc agaaatcatc cacaagaacc agttaacttc 540 agagccattt tcgaacacga attattcggt gttgctttga aacaagcctt cggtaaagat 600 gtcgaatcca tctatgtaaa agaattgggt gtcaccttgt ccagagatga aattttcaag 660 gttttggtcc acgacatgat ggaaggtgct attgatgttg attggagaga tttcttccca 720 tacttgaaat ggatcccaaa caactctttc gaagccagaa ttcaacaaaa gcacaagaga 780 agattggctg ttatgaacgc cttgatccaa gacagattga atcaaaacga ttccgaatcc 840 gatgatgact gctacttgaa tttcttgatg tctgaagcta agaccttgac catggaacaa 900 attgctattt tggtttggga aaccattatc gaaactgctg ataccacttt ggttactact 960 gaatgggcta tgtacgaatt ggccaaacat caatctgttc aagatagatt attcaaagaa 1020 atccaatccg tctgcggtgg tgaaaagatc aaagaagaac aattgccaag attgccttac 1080 gtcaatggtg tttttcacga aaccttgaga aagtattctc cagctccatt ggttccaatt 1140 agatacgctc atgaagatac ccaaattggt ggttatcata ttccagccgg ttctgaaatt 1200 gccattaaca tctacggttg caacatggat aagaagagat gggaaagacc tgaagaatgg 1260 tggccagaaa gatttttgga agatagatac gaatcctccg acttgcataa gactatggct 1320 tttggtgctg gtaaaagagt ttgtgctggt gctttacaag ctagtttgat ggctggtatt 1380 gctatcggta gattggttca agaattcgaa tggaagttga gagatggtga agaagaaaac 1440 gttgatactt acggtttgac ctcccaaaag ttgtatccat tgatggccat tatcaaccca 1500 agaagatctt aa 1512
SEQ ID NO:68
Thellungiella halophila
MASMISLLLG FWSSFLFIF FLKKLLFFFS RHKMSEVSRL PSVPVPGFPL IGNLLQLKEK 60
KPHKTFTKWS ELYGPIYSIK MGSSSLIVLN SIETAKEAMV SRFSSISTRK LSNALTVLTC 120
NKSMVATSDY DDFHKFVKRC LLNGLLGANA QERKRHYRDA LIENVTSKLH AHTRNHPQEP 180
VNFRAIFEHE LFGVALKQAF GKDVESIYVK ELGVTLSRDE IFKVLVHDMM EGAIDVDWRD 240
FFPYLKWIPN NSFEARIQQK HKRRLAVMNA LIQDRLNQND SESDDDCYLN FLMSEAKTLT 300
MEQIAILVWE TIIETADTTL VTTEWAMYEL AKHQSVQDRL FKEIQSVCGG EKIKEEQLPR 360
LPYVNGVFHE TLRKYSPAPL VPIRYAHEDT QIGGYHIPAG SEIAINIYGC NMDKKRWERP 420
EEWWPERFLE DRYESSDLHK TMAFGAGKRV CAGALQASLM AGIAIGRLVQ EFEWKLRDGE 480
EENVDTYGLT SQKLYPLMAI INPRRS 506 SEQ ID NO:69
aagcttacta gtaaaatgga catgatgggt attgaagctg ttccatttgc tactgctgtt 60 gttttgggtg gtatttcctt ggttgttttg atcttcatca gaagattcgt ttccaacaga 120 aagagatccg ttgaaggttt gccaccagtt ccagatattc caggtttacc attgattggt 180 aacttgttgc aattgaaaga aaagaagcca cataagacct ttgctagatg ggctgaaact 240 tacggtccaa ttttctctat tagaactggt gcttctacca tgatcgtctt gaattcttct 300 gaagttgcca aagaagctat ggtcactaga ttctcttcaa tctctaccag aaagttgtcc 360 aacgccttga agattttgac cttcgataag tgtatggttg ccacctctga ttacaacgat 420 tttcacaaaa tggtcaaggg tttcatcttg agaaacgttt taggtgctcc agcccaaaaa 480 agacatagat gtcatagaga taccttgatc gaaaacatct ctaagtactt gcatgcccat 540 gttaagactt ctccattgga accagttgtc ttgaagaaga ttttcgaatc cgaaattttc 600 ggtttggctt tgaaacaagc cttgggtaag gatatcgaat ccatctatgt tgaagaattg 660 ggtactacct tgtccagaga agaaattttt gccgttttgg ttgttgatcc aatggctggt 720 gctattgaag ttgattggag agattttttc ccatacttgt cctggattcc aaacaagtct 780 atggaaatga agatccaaag aatggatttt agaagaggtg ctttgatgaa ggccttgatt 840 ggtgaacaaa agaaaagaat cggttccggt gaagaaaaga actcctacat tgatttcttg 900 ttgtctgaag ctaccacttt gaccgaaaag caaattgcta tgttgatctg ggaaaccatc 960 atcgaaattt ccgatacaac tttggttacc tctgaatggg ctatgtacga attggctaaa 1020 gacccaaata gacaagaaat cttgtacaga gaaatccaca aggtttgcgg ttctaacaag 1080 ttgactgaag aaaacttgtc caagttgcca tacttgaact ctgttttcca cgaaaccttg 1140 agaaagtatt ctccagctcc aatggttcca gttagatatg ctcatgaaga tactcaattg 1200 ggtggttacc atattccagc tggttctcaa attgccatta acatctacgg ttgcaacatg 1260 aacaaaaagc aatgggaaaa tcctgaagaa tggaagccag aaagattctt ggacgaaaag 1320 tatgacttga tggacttgca taagactatg gcttttggtg gtggtaaaag agtttgtgct 1380 ggtgctttac aagcaatgtt gattgcttgc acttccatcg gtagattcgt tcaagaattt 1440 gaatggaagt tgatgggtgg tgaagaagaa aacgttgata ctgttgcttt gacctcccaa 1500 aaattgcatc caatgcaagc cattattaag gccagagaat gactcgagcc gcgg 1554
SEQ ID NO:70
Vitis vinifera
MDMMGIEAVP FATAWLGGI SLWLIFIRR FVSNRKRSVE GLPPVPDIPG LPLIGNLLQL 60
KEKKPHKTFA RWAETYGPIF SIRTGASTMI VLNSSEVAKE AMVTRFSSIS TRKLSNALKI 120
LTFDKCMVAT SDYNDFHKMV KGFILRNVLG APAQKRHRCH RDTLIENISK YLHAHVKTSP 180
LEPWLKKIF ESEIFGLALK QALGKDIESI YVEELGTTLS REEIFAVLW DPMAGAIEVD 240
WRDFFPYLSW IPNKSMEMKI QRMDFRRGAL MKALIGEQKK RIGSGEEKNS YIDFLLSEAT 300
TLTEKQIAML IWETIIEISD TTLVTSEWAM YELAKDPNRQ EILYREIHKV CGSNKLTEEN 360
LSKLPYLNSV FHETLRKYSP APMVPVRYAH EDTQLGGYHI PAGSQIAINI YGCNMNKKQW 420
ENPEEWKPER FLDEKYDLMD LHKTMAFGGG KRVCAGALQA MLIACTSIGR FVQEFEWKLM 480
GGEEENVDTV ALTSQKLHPM QAIIKARE 508
SEQ ID NO:71
aagcttaaaa tgagtaagtc taatagtatg aattctacat cacacgaaac cctttttcaa 60 caattggtct tgggtttgga ccgtatgcca ttgatggatg ttcactggtt gatctacgtt 120 gctttcggcg catggttatg ttcttatgtg atacatgttt tatcatcttc ctctacagta 180 aaagtgccag ttgttggata caggtctgta ttcgaaccta catggttgct tagacttaga 240 ttcgtctggg aaggtggctc tatcataggt caagggtaca ataagtttaa agactctatt 300 ttccaagtta ggaaattggg aactgatatt gtcattatac cacctaacta tattgatgaa 360 gtgagaaaat tgtcacagga caagactaga tcagttgaac ctttcattaa tgattttgca 420 ggtcaataca caagaggcat ggttttcttg caatctgact tacaaaaccg tgttatacaa 480 caaagactaa ctccaaaatt ggtttccttg accaaggtca tgaaggaaga gttggattat 540 gctttaacaa aagagatgcc tgatatgaaa aatgacgaat gggtagaagt agatatcagt 600 agtataatgg tgagattgat ttccaggatc tccgccagag tctttctagg gcctgaacac 660 tgtcgtaacc aggaatggtt gactactaca gcagaatatt cagaatcact tttcattaca 720 gggtttatct taagagttgt acctcatatc ttaagaccat tcatcgcccc tctattacct 780 tcatacagga ctctacttag aaacgtttca agtggtagaa gagtcatcgg tgacatcata 840 agatctcagc aaggggatgg taacgaagat atactttcct ggatgagaga tgctgccaca 900 ggagaggaaa agcaaatcga taacattgct cagagaatgt taattctttc tttagcatca 960 atccacacta ctgcgatgac catgacacat gccatgtacg atctatgtgc ttgccctgag 1020 tacattgaac cattaagaga tgaagttaaa tctgttgttg gggcttctgg ctgggacaag 1080 acagcgttaa acagatttca taagttggac tccttcctaa aagagtcaca aagattcaac 1140 ccagtattct tattgacatt caatagaatc taccatcaat ctatgacctt atcagatggc 1200 actaacattc catctggaac acgtattgct gttccatcac acgcaatgtt gcaagattct 1260 gcacatgtcc caggtccaac cccacctact gaatttgatg gattcagata tagtaagata 1320 cgttctgata gtaactacgc acaaaagtac ctattctcca tgaccgattc ttcaaacatg 1380 gctttcggat acggcaagta tgcttgtcca ggtagatttt acgcgtctaa tgagatgaaa 1440 ctaacattag ccattttgtt gctacaattt gagttcaaac taccagatgg taaaggtcgt 1500 cctagaaata tcactatcga ttctgatatg attccagacc caagagctag actttgcgtc 1560 agaaaaagat cacttagaga tgaatgaccg egg 1593
SEQ ID NO:72
Gibberella fujikuroi
MSKSNSMNST SHETLFQQLV LGLDRMPLMD VHWLIYVAFG AWLCSYVIHV LSSSSTVKVP 60
WGYRSVFEP TWLLRLRFVW EGGSIIGQGY NKFKDSIFQV RKLGTDIVI I PPNYIDEVRK 120
LSQDKTRSVE PFINDFAGQY TRGMVFLQSD LQNRVIQQRL TPKLVSLTKV MKEELDYALT 180
KEMPDMKNDE WVEVDISSIM VRLISRISAR VFLGPEHCRN QEWLTTTAEY SESLFITGFI 240
LRWPHILRP FIAPLLPSYR TLLRNVSSGR RVIGDIIRSQ QGDGNEDILS WMRDAATGEE 300
KQIDNIAQRM LILSLASIHT TAMTMTHAMY DLCACPEYIE PLRDEVKSW GASGWDKTAL 360
NRFHKLDSFL KESQRFNPVF LLTFNRIYHQ SMTLSDGTNI PSGTRIAVPS HAMLQDSAHV 420
PGPTPPTEFD GFRYSKIRSD SNYAQKYLFS MTDSSNMAFG YGKYACPGRF YASNEMKLTL 480
AILLLQFEFK LPDGKGRPRN ITIDSDMIPD PRARLCVRKR SLRDE 525
SEQ ID NO:73
aagcttaaaa tggaagatcc tactgtctta tatgcttgtc ttgccattgc agttgcaact 60 ttcgttgtta gatggtacag agatccattg agatccatcc caacagttgg tggttccgat 120 ttgectatte tatcttacat cggcgcacta agatggacaa gacgtggcag agagatactt 180 caagagggat atgatggcta cagaggatct acattcaaaa tcgcgatgtt agaccgttgg 240 atcgtgatcg caaatggtcc taaactagct gatgaagtca gacgtagacc agatgaagag 300 ttaaacttta tggaeggatt aggagcattc gtccaaacta agtacacctt aggtgaagct 360 attcataacg atccatacca tgtcgatatc ataagagaaa aactaacaag aggccttcca 420 gccgtgcttc ctgatgtcat tgaagagttg acacttgegg ttagacagta cattccaaca 480 gaaggtgatg aatgggtgtc cgtaaactgt teaaaggecg caagagatat tgttgctaga 540 gcttctaata gagtctttgt aggtttgcct gcttgcagaa accaaggtta cttagatttg 600 gcaatagact ttacattgtc tgttgtcaag gatagageca tcatcaatat gtttccagaa 660 ttgttgaagc caatagttgg cagagttgta ggtaacgeca ccagaaatgt tegtagaget 720 gttccttttg ttgetccatt ggtggaggaa agaegtagae ttatggaaga gtacggtgaa 780 gactggtctg aaaaacctaa tgatatgtta cagtggataa tggatgaagc tgcatccaga 840 gatagttcag tgaaggcaat cgcagagaga ttgttaatgg tgaacttege ggctattcat 900 acctcatcaa acactatcac tcatgctttg taccaccttg ccgaaatgcc tgaaactttg 960 caaccactta gagaagagat cgaaccatta gtcaaagagg agggctggac caaggctget 1020 atgggaaaaa tgtggtggtt agattcattt ctaagagaat ctcaaagata caatggcatt 1080 aacategtat ctttaactag aatggctgac aaagatatta cattgagtga tggcacattt 1140 ttgccaaaag gtactctagt ggccgttcca gegtattcta ctcatagaga tgatgctgtc 1200 tacgetgatg ccttagtatt cgatcctttc agattctcac gtatgagagc gagagaaggt 1260 gaaggtacaa agcaccagtt cgttaatact tcagtcgagt aegttccatt tggtcacgga 1320 aagcatgett gtccaggaag attcttcgcc gcaaacgaat tgaaagcaat gttggcttac 1380 attgttctaa actatgatgt aaagttgcct ggtgacggta aacgtccatt gaacatgtat 1440 tggggtccaa cagttttgee tgcaccagca ggecaagtat tgttcagaaa gagacaagtt 1500 agtctataac cgegg 1515
SEQ ID NO:74 Trametes versicolor
MEDPTVLYAC LAIAVATFVV RWYRDPLRSI PTVGGSDLPI LSYIGALRWT RRGREILQEG 60
YDGYRGSTFK IAMLDRWIVI ANGPKLADEV RRRPDEELNF MDGLGAFVQT KYTLGEAIHN 120
DPYHVDIIRE KLTRGLPAVL PDVIEELTLA VRQYI PTEGD EWVSVNCSKA ARDIVARASN 180
RVFVGLPACR NQGYLDLAID FTLSWKDRA IINMFPELLK PIVGRWGNA TRNVRRAVPF 240
VAPLVEERRR LMEEYGEDWS EKPNDMLQWI MDEAASRDSS VKAIAERLLM VNFAAIHTSS 300
NTITHALYHL AEMPETLQPL REEIEPLVKE EGWTKAAMGK MWWLDSFLRE SQRYNGINIV 360
SLTRMADKDI TLSDGTFLPK GTLVAVPAYS THRDDAVYAD ALVFDPFRFS RMRAREGEGT 420
KHQFVNTSVE YVPFGHGKHA CPGRFFAANE LKAMLAYIVL NYDVKLPGDG KRPLNMYWGP 480
TVLPAPAGQV LFRKRQVSL 499
SEQ ID NO:75
atggcatttt tctctatgat ttcaattttg ttgggatttg ttatttcttc tttcatcttc 60 atctttttct tcaaaaagtt acttagtttt agtaggaaaa acatgtcaga agtttctact 120 ttgccaagtg ttccagtagt gcctggtttt ccagttattg ggaatttgtt gcaactaaag 180 gagaaaaagc ctcataaaac tttcactaga tggtcagaga tatatggacc tatctactct 240 ataaagatgg gttcttcatc tcttattgta ttgaacagta cagaaactgc taaggaagca 300 atggtcacta gattttcatc aatatctacc agaaaattgt caaacgccct aacagttcta 360 acctgcgata agtctatggt cgccacttct gattatgatg acttccacaa attagttaag 420 agatgtttgc taaatggact tcttggtgct aatgctcaaa agagaaaaag acactacaga 480 gatgctttga ttgaaaatgt gagttccaag ctacatgcac acgctagaga tcatccacaa 540 gagccagtta actttagagc aattttcgaa cacgaattgt ttggtgtagc attaaagcaa 600 gccttcggta aagacgtaga atccatatac gtcaaggagt taggcgtaac attatcaaaa 660 gatgaaatct ttaaggtgct tgtacatgat atgatggagg gtgcaattga tgtagattgg 720 agagatttct tcccatattt gaaatggatc cctaataagt cttttgaagc taggatacaa 780 caaaagcaca agagaagact agctgttatg aacgcactta tacaggacag attgaagcaa 840 aatgggtctg aatcagatga tgattgttac cttaacttct taatgtctga ggctaaaaca 900 ttgactaagg aacagatcgc aatccttgtc tgggaaacaa tcattgaaac agcagatact 960 accttagtca caactgaatg ggccatatac gagctagcca aacatccatc tgtgcaagat 1020 aggttgtgta aggagatcca gaacgtgtgt ggtggagaga aattcaagga agagcagttg 1080 tcacaagttc cttaccttaa cggcgttttc catgaaacct tgagaaaata ctcacctgca 1140 ccattagttc ctattagata cgcccacgaa gatacacaaa tcggtggcta ccatgttcca 1200 gctgggtccg aaattgctat aaacatctac gggtgcaaca tggacaaaaa gagatgggaa 1260 agaccagaag attggtggcc agaaagattc ttagatgatg gcaaatatga aacatctgat 1320 ttgcataaaa caatggcttt cggagctggc aaaagagtgt gtgccggtgc tctacaagcc 1380 tccctaatgg ctggtatcgc tattggtaga ttggtccaag agttcgaatg gaaacttaga 1440 gatggtgaag aggaaaatgt cgatacttat gggttaacat ctcaaaagtt atacccacta 1500 atggcaatca tcaatcctag aagatcctaa 1530
SEQ ID NO:76
Arabidopsis thaliana
MAFFSMISIL LGFVISSFIF I FFFKKLLSF SRKNMSEVST LPSVPWPGF PVIGNLLQLK 60
EKKPHKTFTR WSEIYGPIYS IKMGSSSLIV LNSTETAKEA MVTRFSSIST RKLSNALTVL 120
TCDKSMVATS DYDDFHKLVK RCLLNGLLGA NAQKRKRHYR DALIENVSSK LHAHARDHPQ 180
EPVNFRAIFE HELFGVALKQ AFGKDVESIY VKELGVTLSK DEIFKVLVHD MMEGAIDVDW 240
RDFFPYLKWI PNKSFEARIQ QKHKRRLAVM NALIQDRLKQ NGSESDDDCY LNFLMSEAKT 300
LTKEQIAILV WETIIETADT TLVTTEWAIY ELAKHPSVQD RLCKEIQNVC GGEKFKEEQL 360
SQVPYLNGVF HETLRKYSPA PLVPIRYAHE DTQIGGYHVP AGSEIAINIY GCNMDKKRWE 420
RPEDWWPERF LDDGKYETSD LHKTMAFGAG KRVCAGALQA SLMAGIAIGR LVQEFEWKLR 480
DGEEENVDTY GLTSQKLYPL MAIINPRRS 509
SEQ ID NO:77
S. rebaudiana
atgcaatcag attcagtcaa agtctctcca tttgatttgg tttccgctgc tatgaatggc 60 aaggcaatgg aaaagttgaa cgctagtgaa tctgaagatc caacaacatt gcctgcacta 120 aagatgctag ttgaaaatag agaattgttg acactgttca caacttcctt cgcagttctt 180 attgggtgtc ttgtatttct aatgtggaga cgttcatcct ctaaaaagct ggtacaagat 240 ccagttccac aagttatcgt tgtaaagaag aaagagaagg agtcagaggt tgatgacggg 300 aaaaagaaag tttctatttt ctacggcaca caaacaggaa ctgccgaagg ttttgctaaa 360 gcattagtcg aggaagcaaa agtgagatat gaaaagacct ctttcaaggt tatcgatcta 420 gatgactacg ctgcagatga tgatgaatat gaggaaaaac tgaaaaagga atccttagcc 480 ttcttcttct tggccacata cggtgatggt gaacctactg ataatgctgc taacttctac 540 aagtggttca cagaaggcga cgataaaggt gaatggctga aaaagttaca atacggagta 600 tttggtttag gtaacagaca atatgaacat ttcaacaaga tcgctattgt agttgatgat 660 aaacttactg aaatgggagc caaaagatta gtaccagtag gattagggga tgatgatcag 720 tgtatagaag atgacttcac cgcctggaag gaattggtat ggccagaatt ggatcaactt 780 ttaagggacg aagatgatac ttctgtgact accccataca ctgcagccgt attggagtac 840 agagtggttt accatgataa accagcagac tcatatgctg aagatcaaac ccatacaaac 900 ggtcatgttg ttcatgatgc acagcatcct tcaagatcta atgtggcttt caaaaaggaa 960 ctacacacct ctcaatcaga taggtcttgt actcacttag aattcgatat ttctcacaca 1020 ggactgtctt acgaaactgg cgatcacgtt ggcgtttatt ccgagaactt gtccgaagtt 1080 gtcgatgaag cactaaaact gttagggtta tcaccagaca catacttctc agtccatgct 1140 gataaggagg atgggacacc tatcggtggt gcttcactac caccaccttt tcctccttgc 1200 acattgagag acgctctaac cagatacgca gatgtcttat cctcacctaa aaaggtagct 1260 ttgctggcat tggctgctca tgctagtgat cctagtgaag ccgataggtt aaagttcctg 1320 gcttcaccag ccggaaaaga tgaatatgca caatggatcg tcgccaacca acgttctttg 1380 ctagaagtga tgcaaagttt tccatctgcc aagcctccat taggtgtgtt cttcgcagca 1440 gtagctccac gtttacaacc aagatactac tctatcagtt catctcctaa gatgtctcct 1500 aacagaatac atgttacatg tgctttggtg tacgagacta ctccagcagg cagaattcac 1560 agaggattgt gttcaacctg gatgaaaaat gctgtccctt taacagagtc acctgattgc 1620 tctcaagcat ccattttcgt tagaacatca aatttcagac ttccagtgga tccaaaagtt 1680 ccagtcatta tgataggacc aggcactggt cttgccccat tcaggggctt tcttcaagag 1740 agattggcct tgaaggaatc tggtacagaa ttgggttctt ctatcttttt ctttggttgc 1800 cgtaatagaa aagttgactt tatctacgag gacgagctta acaattttgt tgagacagga 1860 gcattgtcag aattgatcgt cgcattttca agagaaggga ctgccaaaga gtacgttcag 1920 cacaagatga gtcaaaaagc ctccgatata tggaaacttc taagtgaagg tgcctatctt 1980 tatgtctgtg gcgatgcaaa gggcatggcc aaggatgtcc atagaactct gcatacaatt 2040 gttcaggaac aagggagtct ggattcttcc aaggctgaat tgtacgtcaa aaacttacag 2100 atgtctggaa gatacttaag agatgtttgg taa 2133
SEQ ID NO:78
S. rebaudiana
MQSDSVKVSP FDLVSAAMNG KAMEKLNASE SEDPTTLPAL KMLVENRELL TLFTTSFAVL 60
IGCLVFLMWR RSSSKKLVQD PVPQVIVVKK KEKESEVDDG KKKVSIFYGT QTGTAEGFAK 120
ALVEEAKVRY EKTSFKVIDL DDYAADDDEY EEKLKKESLA FFFLATYGDG EPTDNAANFY 180
KWFTEGDDKG EWLKKLQYGV FGLGNRQYEH FNKIAIWDD KLTEMGAKRL VPVGLGDDDQ 240
CIEDDFTAWK ELVWPELDQL LRDEDDTSVT TPYTAAVLEY RWYHDKPAD SYAEDQTHTN 300
GHWHDAQHP SRSNVAFKKE LHTSQSDRSC THLEFDISHT GLSYETGDHV GVYSENLSEV 360
VDEALKLLGL SPDTYFSVHA DKEDGTPIGG ASLPPPFPPC TLRDALTRYA DVLSSPKKVA 420
LLALAAHASD PSEADRLKFL ASPAGKDEYA QWIVANQRSL LEVMQSFPSA KPPLGVFFAA 480
VAPRLQPRYY SISSSPKMSP NRIHVTCALV YETTPAGRIH RGLCSTWMKN AVPLTESPDC 540
SQASIFVRTS NFRLPVDPKV PVIMIGPGTG LAPFRGFLQE RLALKESGTE LGSSI FFFGC 600
RNRKVDFIYE DELNNFVETG ALSELIVAFS REGTAKEYVQ HKMSQKASDI WKLLSEGAYL 660
YVCGDAKGMA KDVHRTLHTI VQEQGSLDSS KAELYVKNLQ MSGRYLRDVW 710
SEQ ID NO:79
atgaaggtca gtccattcga attcatgtcc gctattatca agggtagaat ggacccatct 60 aactcctcat ttgaatctac tggtgaagtt gcctccgtta tctttgaaaa cagagaattg 120 gttgccatct tgaccacttc tattgctgtt atgattggtt gcttcgttgt cttgatgtgg 180 agaagagctg gttctagaaa ggttaagaat gtcgaattgc caaagccatt gattgtccat 240 gaaccagaac ctgaagttga agatggtaag aagaaggttt ccatcttctt cggtactcaa 300 actggtactg ctgaaggttt tgctaaggct ttggctgatg aagctaaagc tagatacgaa 360 aaggctacct tcagagttgt tgatttggat gattatgctg ccgatgatga ccaatacgaa 420 gaaaaattga agaacgaatc cttcgccgtt ttcttgttgg ctacttatgg tgatggtgaa 480 cctactgata atgctgctag attttacaag tggttcgccg aaggtaaaga aagaggtgaa 540 tggttgcaaa acttgcacta tgctgttttt ggtttgggta acagacaata cgaacacttc 600 aacaagattg ctaaggttgc cgacgaatta ttggaagctc aaggtggtaa tagattggtt 660 aaggttggtt taggtgatga cgatcaatgc atcgaagatg atttttctgc ttggagagaa 720 tctttgtggc cagaattgga tatgttgttg agagatgaag atgatgctac tactgttact 780 actccatata ctgctgctgt cttggaatac agagttgtct ttcatgattc tgctgatgtt 840 gctgctgaag ataagtcttg gattaacgct aatggtcatg ctgttcatga tgctcaacat 900 ccattcagat ctaacgttgt cgtcagaaaa gaattgcata cttctgcctc tgatagatcc 960 tgttctcatt tggaattcaa catttccggt tccgctttga attacgaaac tggtgatcat 1020 gttggtgtct actgtgaaaa cttgactgaa actgttgatg aagccttgaa cttgttgggt 1080 ttgtctccag aaacttactt ctctatctac accgataacg aagatggtac tccattgggt 1140 ggttcttcat tgccaccacc atttccatca tgtactttga gaactgcttt gaccagatac 1200 gctgatttgt tgaactctcc aaaaaagtct gctttgttgg ctttagctgc tcatgcttct 1260 aatccagttg aagctgatag attgagatac ttggcttctc cagctggtaa agatgaatat 1320 gcccaatctg ttatcggttc ccaaaagtct ttgttggaag ttatggctga attcccatct 1380 gctaaaccac cattaggtgt tttttttgct gctgttgctc caagattgca acctagattc 1440 tactccattt catcctctcc aagaatggct ccatctagaa tccatgttac ttgtgctttg 1500 gtttacgata agatgccaac tggtagaatt cataagggtg tttgttctac ctggatgaag 1560 aattctgttc caatggaaaa gtcccatgaa tgttcttggg ctccaatttt cgttagacaa 1620 tccaatttta agttgccagc cgaatccaag gttccaatta tcatggttgg tccaggtact 1680 ggtttggctc cttttagagg ttttttacaa gaaagattgg ccttgaaaga atccggtgtt 1740 gaattgggtc catccatttt gtttttcggt tgcagaaaca gaagaatgga ttacatctac 1800 gaagatgaat tgaacaactt cgttgaaacc ggtgctttgt ccgaattggt tattgctttt 1860 tctagagaag gtcctaccaa agaatacgtc caacataaga tggctgaaaa ggcttctgat 1920 atctggaact tgatttctga aggtgcttac ttgtacgttt gtggtgatgc taaaggtatg 1980 gctaaggatg ttcatagaac cttgcatacc atcatgcaag aacaaggttc tttggattct 2040 tccaaagctg aatccatggt caagaacttg caaatgaatg gtagatactt aagagatgtt 2100 tggtaa 2106
SEQ ID NO:80
Siraitia grosvenorii
MKVSPFEFMS AIIKGRMDPS NSSFESTGEV ASVIFENREL VAILTTSIAV MIGCFWLMW 60
RRAGSRKVKN VELPKPLIVH EPEPEVEDGK KKVSI FFGTQ TGTAEGFAKA LADEAKARYE 120
KATFRWDLD DYAADDDQYE EKLKNESFAV FLLATYGDGE PTDNAARFYK WFAEGKERGE 180
WLQNLHYAVF GLGNRQYEHF NKIAKVADEL LEAQGGNRLV KVGLGDDDQC IEDDFSAWRE 240
SLWPELDMLL RDEDDATTVT TPYTAAVLEY RVVFHDSADV AAEDKSWINA NGHAVHDAQH 300
PFRSNVWRK ELHTSASDRS CSHLEFNISG SALNYETGDH VGVYCENLTE TVDEALNLLG 360
LSPETYFSIY TDNEDGTPLG GSSLPPPFPS CTLRTALTRY ADLLNSPKKS ALLALAAHAS 420
NPVEADRLRY LASPAGKDEY AQSVIGSQKS LLEVMAEFPS AKPPLGVFFA AVAPRLQPRF 480
YSISSSPRMA PSRIHVTCAL VYDKMPTGRI HKGVCSTWMK NSVPMEKSHE CSWAPIFVRQ 540
SNFKLPAESK VPIIMVGPGT GLAPFRGFLQ ERLALKESGV ELGPSILFFG CRNRRMDYIY 600
EDELNNFVET GALSELVIAF SREGPTKEYV QHKMAEKASD IWNLISEGAY LYVCGDAKGM 660
AKDVHRTLHT IMQEQGSLDS SKAESMVKNL QMNGRYLRDV W 701
SEQ ID NO:81
atggcagaat tagatacact tgatatagta gtattaggtg ttatcttttt gggtactgtg 60 gcatacttta ctaagggtaa attgtggggt gttaccaagg atccatacgc taacggattc 120 gctgcaggtg gtgcttccaa gcctggcaga actagaaaca tcgtcgaagc tatggaggaa 180 tcaggtaaaa actgtgttgt tttctacggc agtcaaacag gtacagcgga ggattacgca 240 tcaagacttg caaaggaagg aaagtccaga ttcggtttga acactatgat cgccgatcta 300 gaagattatg acttcgataa cttagacact gttccatctg ataacatcgt tatgtttgta 360 ttggctactt acggtgaagg cgaaccaaca gataacgccg tggatttcta tgagttcatt 420 actggcgaag atgcctcttt caatgagggc aacgatcctc cactaggtaa cttgaattac 480 gttgcgttcg gtctgggcaa caatacctac gaacactaca actcaatggt caggaacgtt 540 aacaaggctc tagaaaagtt aggagctcat agaattggag aagcaggtga gggtgacgac 600 ggagctggaa ctatggaaga ggacttttta gcttggaaag atccaatgtg ggaagccttg 660 gctaaaaaga tgggcttgga ggaaagagaa gctgtatatg aacctatttt cgctatcaat 720 gagagagatg atttgacccc tgaagcgaat gaggtatact tgggagaacc taataagcta 780 cacttggaag gtacagcgaa aggtccattc aactcccaca acccatatat cgcaccaatt 840 gcagaatcat acgaactttt ctcagctaag gatagaaatt gtctgcatat ggaaattgat 900 atttctggta gtaatctaaa gtatgaaaca ggcgaccata tcgcgatctg gcctaccaac 960 ccaggtgaag aggtcaacaa atttcttgac attctagatc tgtctggtaa gcaacattcc 1020 gtcgtaacag tgaaagcctt agaacctaca gccaaagttc cttttccaaa tccaactacc 1080 tacgatgcta tattgagata ccatctggaa atatgcgctc cagtttctag acagtttgtc 1140 tcaactttag cagcattcgc ccctaatgat gatatcaaag ctgagatgaa ccgtttggga 1200 tcagacaaag attacttcca cgaaaagaca ggaccacatt actacaatat cgctagattt 1260 ttggcctcag tctctaaagg tgaaaaatgg acaaagatac cattttctgc tttcatagaa 1320 ggccttacaa aactacaacc aagatactat tctatctctt cctctagttt agttcagcct 1380 aaaaagatta gtattactgc tgttgtcgaa tctcagcaaa ttccaggtag agatgaccca 1440 ttcagaggtg tagcgactaa ctacttgttc gctttgaagc agaaacaaaa cggtgatcca 1500 aatccagctc cttttggcca atcatacgag ttgacaggac caaggaataa gtatgatggt 1560 atacatgttc cagtccatgt aagacattct aactttaagc taccatctga tccaggcaaa 1620 cctattatca tgatcggtcc aggtaccggt gttgcccctt ttagaggctt cgtccaagag 1680 agggcaaaac aagccagaga tggtgtagaa gttggtaaaa cactgctgtt ctttggatgt 1740 agaaagagta cagaagattt catgtatcaa aaagagtggc aagagtacaa ggaagctctt 1800 ggcgacaaat tcgaaatgat tacagctttt tcaagagaag gatctaaaaa ggtttatgtt 1860 caacacagac tgaaggaaag atcaaaggaa gtttctgatc ttctatccca aaaagcatac 1920 ttctacgttt gcggagacgc cgcacatatg gcacgtgaag tgaacactgt gttagcacag 1980 atcatagcag aaggccgtgg tgtatcagaa gccaagggtg aggaaattgt caaaaacatg 2040 agatcagcaa atcaatacca agtgtgttct gatttcgtaa ctttacactg taaagagaca 2100 acatacgcga attcagaatt gcaagaggat gtctggagtt aa 2142
SEQ ID NO:82
Gibberella fujikuroi
MAELDTLDIV VLGVIFLGTV AYFTKGKLWG VTKDPYANGF AAGGASKPGR TRNIVEAMEE 60
SGKNCWFYG SQTGTAEDYA SRLAKEGKSR FGLNTMIADL EDYDFDNLDT VPSDNIVMFV 120
LATYGEGEPT DNAVDFYEFI TGEDASFNEG NDPPLGNLNY VAFGLGNNTY EHYNSMVRNV 180
NKALEKLGAH RIGEAGEGDD GAGTMEEDFL AWKDPMWEAL AKKMGLEERE AVYEPIFAIN 240
ERDDLTPEAN EVYLGEPNKL HLEGTAKGPF NSHNPYIAPI AESYELFSAK DRNCLHMEID 300
ISGSNLKYET GDHIAIWPTN PGEEVNKFLD ILDLSGKQHS WTVKALEPT AKVPFPNPTT 360
YDAILRYHLE ICAPVSRQFV STLAAFAPND DIKAEMNRLG SDKDYFHEKT GPHYYNIARF 420
LASVSKGEKW TKI PFSAFIE GLTKLQPRYY SISSSSLVQP KKISITAWE SQQIPGRDDP 480
FRGVATNYLF ALKQKQNGDP NPAPFGQSYE LTGPRNKYDG IHVPVHVRHS NFKLPSDPGK 540
PIIMIGPGTG VAPFRGFVQE RAKQARDGVE VGKTLLFFGC RKSTEDFMYQ KEWQEYKEAL 600
GDKFEMITAF SREGSKKVYV QHRLKERSKE VSDLLSQKAY FYVCGDAAHM AREVNTVLAQ 660
IIAEGRGVSE AKGEEIVKNM RSANQYQVCS DFVTLHCKET TYANSELQED VWS 713
SEQ ID NO:83
atgcaatcgg aatccgttga agcatcgacg attgatttga tgactgctgt tttgaaggac 60 acagtgatcg atacagcgaa cgcatctgat aacggagact caaagatgcc gccggcgttg 120 gcgatgatgt tcgaaattcg tgatctgttg ctgattttga ctacgtcagt tgctgttttg 180 gtcggatgtt tcgttgtttt ggtgtggaag agatcgtccg ggaagaagtc cggcaaggaa 240 ttggagccgc cgaagatcgt tgtgccgaag aggcggctgg agcaggaggt tgatgatggt 300 aagaagaagg ttacgatttt cttcggaaca caaactggaa cggctgaagg tttcgctaag 360 gcacttttcg aagaagcgaa agcgcgatat gaaaaggcag cgtttaaagt gattgatttg 420 gatgattatg ctgctgattt ggatgagtat gcagagaagc tgaagaagga aacatatgct 480 ttcttcttct tggctacata tggagatggt gagccaactg ataatgctgc caaattttat 540 aaatggttta ctgagggaga cgagaaaggc gtttggcttc aaaaacttca atatggagta 600 tttggtcttg gcaacagaca atatgaacat ttcaacaaga ttggaatagt ggttgatgat 660 ggtctcaccg agcagggtgc aaaacgcatt gttcccgttg gtcttggaga cgacgatcaa 720 tcaattgaag acgatttttc ggcatggaaa gagttagtgt ggcccgaatt ggatctattg 780 cttcgcgatg aagatgacaa agctgctgca actccttaca cagctgcaat ccctgaatac 840 cgcgtcgtat ttcatgacaa acccgatgcg ttttctgatg atcatactca aaccaatggt 900 catgctgttc atgatgctca acatccatgc agatccaatg tggctgttaa aaaagagctt 960 catactcctg aatccgatcg ttcatgcaca catcttgaat ttgacatttc tcacactgga 1020 ttatcttatg aaactgggga tcatgttggt gtatactgtg aaaacctaat tgaagtagtg 1080 gaagaagctg ggaaattgtt aggattatca acagatactt atttctcgtt acatattgat 1140 aacgaagatg gttcaccact tggtggacct tcattacaac ctccttttcc tccttgtact 1200 ttaagaaaag cattgactaa ttatgcagat ctgttaagct ctcccaaaaa gtcaactttg 1260 cttgctctag ctgctcatgc ttccgatccc actgaagctg atcgtttaag atttcttgca 1320 tctcgcgagg gcaaggatga atatgctgaa tgggttgttg caaaccaaag aagtcttctt 1380 gaagtcatgg aagctttccc gtcagctaga ccgccacttg gtgttttctt tgcagcggtt 1440 gcaccgcgtt tacagcctcg ttactactct atttcttcct ccccaaagat ggaaccaaac 1500 aggattcatg ttacttgcgc gttggtttat gaaaaaactc ccgcaggtcg tatccacaaa 1560 ggaatctgct caacctggat gaagaacgct gtacctttga ccgaaagtca agattgcagt 1620 tgggcaccga tttttgttag aacatcaaac ttcagacttc caattgaccc gaaagtcccg 1680 gttatcatga ttggtcctgg aaccgggttg gctccattta ggggttttct tcaagaaaga 1740 ttggctctta aagaatccgg aaccgaactc gggtcatcta ttttattctt cggttgtaga 1800 aaccgcaaag tggattacat atatgagaat gaactcaaca actttgttga aaatggtgcg 1860 ctttctgagc ttgatgttgc tttctcccgc gatggcccga cgaaagaata cgtgcaacat 1920 aaaatgaccc aaaaggcttc tgaaatatgg aatatgcttt ctgagggagc atatttatat 1980 gtatgtggtg atgctaaagg catggctaaa gatgtacacc gtacacttca caccattgtg 2040 caagaacagg gaagtttgga ctcgtctaaa gcggagttgt atgtgaagaa tctacaaatg 2100 tcaggaagat acctccgtga tgtttggtaa 2130
SEQ ID NO:84
Stevia rebaudiana
MQSESVEAST IDLMTAVLKD TVIDTANASD NGDSKMPPAL AMMFEIRDLL LILTTSVAVL 60 VGCFVVLVWK RSSGKKSGKE LEPPKIVVPK RRLEQEVDDG KKKVTIFFGT QTGTAEGFAK 120 ALFEEAKARY EKAAFKVIDL DDYAADLDEY AEKLKKETYA FFFLATYGDG EPTDNAAKFY 180 KWFTEGDEKG VWLQKLQYGV FGLGNRQYEH FNKIGIWDD GLTEQGAKRI VPVGLGDDDQ 240 SIEDDFSAWK ELVWPELDLL LRDEDDKAAA TPYTAAIPEY RWFHDKPDA FSDDHTQTNG 300 HAVHDAQHPC RSNVAVKKEL HTPESDRSCT HLEFDISHTG LSYETGDHVG VYCENLIEW 360 EEAGKLLGLS TDTYFSLHID NEDGSPLGGP SLQPPFPPCT LRKALTNYAD LLSSPKKSTL 420 LALAAHASDP TEADRLRFLA SREGKDEYAE WVVANQRSLL EVMEAFPSAR PPLGVFFAAV 480 APRLQPRYYS ISSSPKMEPN RIHVTCALVY EKTPAGRIHK GICSTWMKNA VPLTESQDCS 540 WAPIFVRTSN FRLPIDPKVP VIMIGPGTGL APFRGFLQER LALKESGTEL GSSILFFGCR 600 NRKVDYIYEN ELNNFVENGA LSELDVAFSR DGPTKEYVQH KMTQKASEIW NMLSEGAYLY 660 VCGDAKGMAK DVHRTLHTIV QEQGSLDSSK AELYVKNLQM SGRYLRDVW 709
SEQ ID NO:85
S. rebaudiana
atgcaatcta actccgtgaa gatttcgccg cttgatctgg taactgcgct gtttagcggc 60 aaggttttgg acacatcgaa cgcatcggaa tcgggagaat ctgctatgct gccgactata 120 gcgatgatta tggagaatcg tgagctgttg atgatactca caacgtcggt tgctgtattg 180 atcggatgcg ttgtcgtttt ggtgtggcgg agatcgtcta cgaagaagtc ggcgttggag 240 ccaccggtga ttgtggttcc gaagagagtg caagaggagg aagttgatga tggtaagaag 300 aaagttacgg ttttcttcgg cacccaaact ggaacagctg aaggcttcgc taaggcactt 360 gttgaggaag ctaaagctcg atatgaaaag gctgtcttta aagtaattga tttggatgat 420 tatgctgctg atgacgatga gtatgaggag aaactaaaga aagaatcttt ggcctttttc 480 tttttggcta cgtatggaga tggtgagcca acagataatg ctgccagatt ttataaatgg 540 tttactgagg gagatgcgaa aggagaatgg cttaataagc ttcaatatgg agtatttggt 600 ttgggtaaca gacaatatga acattttaac aagatcgcaa aagtggttga tgatggtctt 660 gtagaacagg gtgcaaagcg tcttgttcct gttggacttg gagatgatga tcaatgtatt 720 gaagatgact tcaccgcatg gaaagagtta gtatggccgg agttggatca attacttcgt 780 gatgaggatg acacaactgt tgctactcca tacacagctg ctgttgcaga atatcgcgtt 840 gtttttcatg aaaaaccaga cgcgctttct gaagattata gttatacaaa tggccatgct 900 gttcatgatg ctcaacatcc atgcagatcc aacgtggctg tcaaaaagga acttcatagt 960 cctgaatctg accggtcttg cactcatctt gaatttgaca tctcgaacac cggactatca 1020 tatgaaactg gggaccatgt tggagtttac tgtgaaaact tgagtgaagt tgtgaatgat 1080 gctgaaagat tagtaggatt accaccagac acttactcct ccatccacac tgatagtgaa 1140 gacgggtcgc cacttggcgg agcctcattg ccgcctcctt tcccgccatg cactttaagg 1200 aaagcattga cgtgttatgc tgatgttttg agttctccca agaagtcggc tttgcttgca 1260 ctagctgctc atgccaccga tcccagtgaa gctgatagat tgaaatttct tgcatccccc 1320 gccggaaagg atgaatattc tcaatggata gttgcaagcc aaagaagtct ccttgaagtc 1380 atggaagcat tcccgtcagc taagccttca cttggtgttt tctttgcatc tgttgccccg 1440 cgcttacaac caagatacta ctctatttct tcctcaccca agatggcacc ggataggatt 1500 catgttacat gtgcattagt ctatgagaaa acacctgcag gccgcatcca caaaggagtt 1560 tgttcaactt ggatgaagaa cgcagtgcct atgaccgaga gtcaagattg cagttgggcc 1620 ccaatatacg tccgaacatc caatttcaga ctaccatctg accctaaggt cccggttatc 1680 atgattggac ctggcactgg tttggctcct tttagaggtt tccttcaaga gcggttagct 1740 ttaaaggaag ccggaactga cctcggttta tccattttat tcttcggatg taggaatcgc 1800 aaagtggatt tcatatatga aaacgagctt aacaactttg tggagactgg tgctctttct 1860 gagcttattg ttgctttctc ccgtgaaggc ccgactaagg aatatgtgca acacaagatg 1920 agtgagaagg cttcggatat ctggaacttg ctttctgaag gagcatattt atacgtatgt 1980 ggtgatgcca aaggcatggc caaagatgta catcgaaccc tccacacaat tgtgcaagaa 2040 cagggatctc ttgactcgtc aaaggcagaa ctctacgtga agaatctaca aatgtcagga 2100 agatacctcc gtgacgtttg gtaa 2124
SEQ ID NO:86
S. rebaudiana
MQSNSVKISP LDLVTALFSG KVLDTSNASE SGESAMLPTI AMIMENRELL MILTTSVAVL 60
IGCWVLVWR RSSTKKSALE PPVIWPKRV QEEEVDDGKK KVTVFFGTQT GTAEGFAKAL 120
VEEAKARYEK AVFKVIDLDD YAADDDEYEE KLKKESLAFF FLATYGDGEP TDNAARFYKW 180
FTEGDAKGEW LNKLQYGVFG LGNRQYEHFN KIAKVVDDGL VEQGAKRLVP VGLGDDDQCI 240
EDDFTAWKEL VWPELDQLLR DEDDTTVATP YTAAVAEYRV VFHEKPDALS EDYSYTNGHA 300
VHDAQHPCRS NVAVKKELHS PESDRSCTHL EFDISNTGLS YETGDHVGVY CENLSEVVND 360
AERLVGLPPD TYSSIHTDSE DGSPLGGASL PPPFPPCTLR KALTCYADVL SSPKKSALLA 420
LAAHATDPSE ADRLKFLASP AGKDEYSQWI VASQRSLLEV MEAFPSAKPS LGVFFASVAP 480
RLQPRYYSIS SSPKMAPDRI HVTCALVYEK TPAGRIHKGV CSTWMKNAVP MTESQDCSWA 540
PIYVRTSNFR LPSDPKVPVI MIGPGTGLAP FRGFLQERLA LKEAGTDLGL SILFFGCRNR 600
KVDFIYENEL NNFVETGALS ELIVAFSREG PTKEYVQHKM SEKASDIWNL LSEGAYLYVC 660
GDAKGMAKDV HRTLHTIVQE QGSLDSSKAE LYVKNLQMSG RYLRDVW 707
SEQ ID NO:87
atgtcctcca actccgattt ggtcagaaga ttggaatctg ttttgggtgt ttctttcggt 60 ggttctgtta ctgattccgt tgttgttatt gctaccacct ctattgcttt ggttatcggt 120 gttttggttt tgttgtggag aagatcctct gacagatcta gagaagttaa gcaattggct 180 gttccaaagc cagttactat cgttgaagaa gaagatgaat tcgaagttgc ttctggtaag 240 accagagttt ctattttcta cggtactcaa actggtactg ctgaaggttt tgctaaggct 300 ttggctgaag aaatcaaagc cagatacgaa aaagctgccg ttaaggttat tgatttggat 360 gattacacag ccgaagatga caaatacggt gaaaagttga agaaagaaac tatggccttc 420 ttcatgttgg ctacttatgg tgatggtgaa cctactgata atgctgctag attttacaag 480 tggttcaccg aaggtactga tagaggtgtt tggttggaac atttgagata cggtgtattc 540 ggtttgggta acagacaata cgaacacttc aacaagattg ccaaggttgt tgatgatttg 600 ttggttgaac aaggtgccaa gagattggtt actgttggtt tgggtgatga tgatcaatgc 660 atcgaagatg atttctccgc ttggaaagaa gccttgtggc cagaattgga tcaattattg 720 caagatgata ccaacaccgt ttctactcca tacactgctg ttattccaga atacagagtt 780 gttatccacg atccatctgt tacctcttat gaagatccat actctaacat ggctaacggt 840 aatgcctctt acgatattca tcatccatgt agagctaacg ttgccgtcca aaaagaattg 900 cataagccag aatctgacag aagttgcatc catttggaat tcgatatttt cgctactggt 960 ttgacttacg aaaccggtga tcatgttggt gtttacgctg ataattgtga tgatactgta 1020 gaagaagccg ctaagttgtt gggtcaacca ttggatttgt tgttctccat tcataccgat 1080 aacaacgacg gtacttcttt gggttcttct ttgccaccac catttccagg tccatgtact 1140 ttgagaactg ctttggctag atatgccgat ttgttgaatc caccaaaaaa ggctgctttg 1200 attgctttag ctgctcatgc tgatgaacca tctgaagctg aaagattgaa gttcttgtca 1260 tctccacaag gtaaggacga atattctaaa tgggttgtcg gttcccaaag atccttggtt 1320 gaagttatgg ctgaatttcc atctgctaaa ccaccattgg gtgtattttt tgctgctgtt 1380 gttcctagat tgcaacctag atattactcc atctcttcca gtccaagatt tgctccacat 1440 agagttcatg ttacttgcgc tttggtttat ggtccaactc caactggtag aattcacaga 1500 ggtgtatgtt cattctggat gaagaatgtt gtcccattgg aaaagtctca aaactgttct 1560 tgggccccaa ttttcatcag acaatctaat ttcaagttgc cagccgatca ttctgttcca 1620 atagttatgg ttggtccagg tactggttta gctcctttta gaggtttctt acaagaaaga 1680 ttggccttga aagaagaagg tgctcaagtt ggtcctgctt tgttgttttt tggttgcaga 1740 aacagacaaa tggacttcat ctacgaagtc gaattgaaca actttgtcga acaaggtgct 1800 ttgtccgaat tgatcgttgc tttttcaaga gaaggtccat ccaaagaata cgtccaacat 1860 aagatggttg aaaaggcagc ttacatgtgg aacttgattt ctcaaggtgg ttacttctac 1920 gtttgtggtg atgctaaagg tatggctaga gatgttcata gaacattgca taccatcgtc 1980 caacaagaag aaaaggttga ttctaccaag gccgaatcca tcgttaagaa attgcaaatg 2040 gacggtagat acttgagaga tgtttggtga 2070
SEQ ID NO:88
Rubus suavissimus
MSSNSDLVRR LESVLGVSFG GSVTDSVWI ATTSIALVIG VLVLLWRRSS DRSREVKQLA 60
VPKPVTIVEE EDEFEVASGK TRVSI FYGTQ TGTAEGFAKA LAEEIKARYE KAAVKVIDLD 120
DYTAEDDKYG EKLKKETMAF FMLATYGDGE PTDNAARFYK WFTEGTDRGV WLEHLRYGVF 180
GLGNRQYEHF NKIAKVVDDL LVEQGAKRLV TVGLGDDDQC IEDDFSAWKE ALWPELDQLL 240
QDDTNTVSTP YTAVIPEYRV VIHDPSVTSY EDPYSNMANG NASYDIHHPC RANVAVQKEL 300
HKPESDRSCI HLEFDIFATG LTYETGDHVG VYADNCDDTV EEAAKLLGQP LDLLFSIHTD 360
NNDGTSLGSS LPPPFPGPCT LRTALARYAD LLNPPKKAAL IALAAHADEP SEAERLKFLS 420
SPQGKDEYSK WWGSQRSLV EVMAEFPSAK PPLGVFFAAV VPRLQPRYYS ISSSPRFAPH 480
RVHVTCALVY GPTPTGRIHR GVCSFWMKNV VPLEKSQNCS WAPIFIRQSN FKLPADHSVP 540
IVMVGPGTGL APFRGFLQER LALKEEGAQV GPALLFFGCR NRQMDFIYEV ELNNFVEQGA 600
LSELIVAFSR EGPSKEYVQH KMVEKAAYMW NLISQGGYFY VCGDAKGMAR DVHRTLHTIV 660
QQEEKVDSTK AESIVKKLQM DGRYLRDVW 689
SEQ ID NO:89
atgacttctg cactttatgc ctccgatctt ttcaaacaat tgaaaagtat catgggaacg 60 gattctttgt ccgatgatgt tgtattagtt attgctacaa cttctctggc actggttgct 120 ggtttcgttg tcttattgtg gaaaaagacc acggcagatc gttccggcga gctaaagcca 180 ctaatgatcc ctaagtctct gatggcgaaa gatgaggatg atgacttaga tctaggttct 240 ggaaaaacga gagtctctat cttcttcggc acacaaaccg gaacagccga aggattcgct 300 aaagcacttt cagaagagat caaagcaaga tacgaaaagg cggctgtaaa agtaatcgat 360 ttggatgatt acgctgccga tgatgaccaa tatgaggaaa agttgaaaaa ggaaacattg 420 gctttctttt gtgtagccac gtatggtgat ggtgaaccaa ccgataacgc cgcaagattc 480 tacaagtggt ttactgaaga gaacgaaaga gatatcaagt tgcagcaact tgcttacggc 540 gtttttgcct taggtaacag acaatacgag cactttaaca agataggtat tgtcttagat 600 gaagagttat gcaaaaaggg tgcgaagaga ttgattgaag tcggtttagg agatgatgat 660 caatctatcg aggatgactt taatgcatgg aaggaatctt tgtggtctga attagataag 720 ttacttaagg acgaagatga taaatccgtt gccactccat acacagccgt cattccagaa 780 tatagagtag ttactcatga tccaagattc acaacacaga aatcaatgga aagtaatgtg 840 gctaatggta atactaccat cgatattcat catccatgta gagtagacgt tgcagttcaa 900 aaggaattgc acactcatga atcagacaga tcttgcatac atcttgaatt tgatatatca 960 cgtactggta tcacttacga aacaggtgat cacgtgggtg tctacgctga aaaccatgtt 1020 gaaattgtag aggaagctgg aaagttgttg ggccatagtt tagatcttgt tttctcaatt 1080 catgccgata aagaggatgg ctcaccacta gaaagtgcag tgcctccacc atttccagga 1140 ccatgcaccc taggtaccgg tttagctcgt tacgcggatc tgttaaatcc tccacgtaaa 1200 tcagctctag tggccttggc tgcgtacgcc acagaacctt ctgaggcaga aaaactgaaa 1260 catctaactt caccagatgg taaggatgaa tactcacaat ggatagtagc tagtcaacgt 1320 tctttactag aagttatggc tgctttccca tccgctaaac ctcctttggg tgttttcttc 1380 gccgcaatag cgcctagact gcaaccaaga tactattcaa tttcatcctc acctagactg 1440 gcaccatcaa gagttcatgt cacatccgct ttagtgtacg gtccaactcc tactggtaga 1500 atccataagg gcgtttgttc aacatggatg aaaaacgcgg ttccagcaga gaagtctcac 1560 gaatgttctg gtgctccaat ctttatcaga gcctccaact tcaaactgcc ttccaatcct 1620 tctactccta ttgtcatggt cggtcctggt acaggtcttg ctccattcag aggtttctta 1680 caagagagaa tggccttaaa ggaggatggt gaagagttgg gatcttcttt gttgtttttc 1740 ggctgtagaa acagacaaat ggatttcatc tacgaagatg aactgaataa ctttgtagat 1800 caaggagtta tttcagagtt gataatggct ttttctagag aaggtgctca gaaggagtac 1860 gtccaacaca aaatgatgga aaaggccgca caagtttggg acttaatcaa agaggaaggc 1920 tatctatatg tctgtggtga tgcaaagggt atggcaagag atgttcacag aacacttcat 1980 actatagtcc aggaacagga aggcgttagt tcttctgaag cggaagcaat tgtgaaaaag 2040 ttacaaacag agggaagata cttgagagat gtgtggtaa 2079
SEQ ID NO:90
Arabidopsis thaliana
MTSALYASDL FKQLKSIMGT DSLSDDVVLV IATTSLALVA GFWLLWKKT TADRSGELKP 60 LMIPKSLMAK DEDDDLDLGS GKTRVSI FFG TQTGTAEGFA KALSEEIKAR YEKAAVKVID 120 LDDYAADDDQ YEEKLKKETL AFFCVATYGD GEPTDNAARF YKWFTEENER DIKLQQLAYG 180 VFALGNRQYE HFNKIGIVLD EELCKKGAKR LIEVGLGDDD QSIEDDFNAW KESLWSELDK 240 LLKDEDDKSV ATPYTAVIPE YRWTHDPRF TTQKSMESNV ANGNTTIDIH HPCRVDVAVQ 300 KELHTHESDR SCIHLEFDIS RTGITYETGD HVGVYAENHV EIVEEAGKLL GHSLDLVFSI 360 HADKEDGSPL ESAVPPPFPG PCTLGTGLAR YADLLNPPRK SALVALAAYA TEPSEAEKLK 420 HLTSPDGKDE YSQWIVASQR SLLEVMAAFP SAKPPLGVFF AAIAPRLQPR YYSISSSPRL 480 APSRVHVTSA LVYGPTPTGR IHKGVCSTWM KNAVPAEKSH ECSGAPIFIR ASNFKLPSNP 540 STPIVMVGPG TGLAPFRGFL QERMALKEDG EELGSSLLFF GCRNRQMDFI YEDELNNFVD 600 QGVISELIMA FSREGAQKEY VQHKMMEKAA QWDLIKEEG YLYVCGDAKG MARDVHRTLH 660 TIVQEQEGVS SSEAEAIVKK LQTEGRYLRD VW 692
SEQ ID NO:91
A. thaliana
atgtcttcct cttcctcttc cagtacctct atgattgatt tgatggctgc tattattaaa 60 ggtgaaccag ttatcgtctc cgacccagca aatgcctctg cttatgaatc agttgctgca 120 gaattgtctt caatgttgat cgaaaacaga caattcgcca tgatcgtaac tacatcaatc 180 gctgttttga tcggttgtat tgtcatgttg gtatggagaa gatccggtag tggtaattct 240 aaaagagtcg aacctttgaa accattagta attaagccaa gagaagaaga aatagatgac 300 ggtagaaaga aagttacaat atttttcggt acccaaactg gtacagctga aggttttgca 360 aaagccttag gtgaagaagc taaggcaaga tacgaaaaga ctagattcaa gatagtcgat 420 ttggatgact atgccgctga tgacgatgaa tacgaagaaa agttgaagaa agaagatgtt 480 gcatttttct ttttggcaac ctatggtgac ggtgaaccaa ctgacaatgc agccagattc 540 tacaaatggt ttacagaggg taatgatcgt ggtgaatggt tgaaaaactt aaagtacggt 600 gttttcggtt tgggtaacag acaatacgaa catttcaaca aagttgcaaa ggttgtcgac 660 gatattttgg tcgaacaagg tgctcaaaga ttagtccaag taggtttggg tgacgatgac 720 caatgtatag aagatgactt tactgcctgg agagaagctt tgtggcctga attagacaca 780 atcttgagag aagaaggtga caccgccgtt gctaccccat atactgctgc agtattagaa 840 tacagagttt ccatccatga tagtgaagac gcaaagttta atgatatcac tttggccaat 900 ggtaacggtt atacagtttt cgatgcacaa cacccttaca aagctaacgt tgcagtcaag 960 agagaattac atacaccaga atccgacaga agttgtatac acttggaatt tgatatcgct 1020 ggttccggtt taaccatgaa gttgggtgac catgtaggtg ttttatgcga caatttgtct 1080 gaaactgttg atgaagcatt gagattgttg gatatgtccc ctgacactta ttttagtttg 1140 cacgctgaaa aagaagatgg tacaccaatt tccagttctt taccacctcc attccctcca 1200 tgtaacttaa gaacagcctt gaccagatac gcttgcttgt tatcatcccc taaaaagtcc 1260 gccttggttg ctttagccgc tcatgctagt gatcctactg aagcagaaag attgaaacac 1320 ttagcatctc cagccggtaa agatgaatat tcaaagtggg tagttgaatc tcaaagatca 1380 ttgttagaag ttatggcaga atttccatct gccaagcctc cattaggtgt cttctttgct 1440 ggtgtagcac ctagattgca accaagattc tactcaatca gttcttcacc taagatcgct 1500 gaaactagaa ttcatgttac atgtgcatta gtctacgaaa agatgccaac cggtagaatt 1560 cacaagggtg tatgctctac ttggatgaaa aatgctgttc cttacgaaaa atcagaaaag 1620 ttgttcttag gtagaccaat cttcgtaaga caatcaaact tcaagttgcc ttctgattca 1680 aaggttccaa taatcatgat aggtcctggt acaggtttag ccccattcag aggtttcttg 1740 caagaaagat tggctttagt tgaatctggt gtcgaattag gtccttcagt tttgttcttt 1800 ggttgtagaa acagaagaat ggatttcatc tatgaagaag aattgcaaag attcgtcgaa 1860 tctggtgcat tggccgaatt atctgtagct ttttcaagag aaggtccaac taaggaatac 1920 gttcaacata agatgatgga taaggcatcc gacatatgga acatgatcag tcaaggtgct 1980 tatttgtacg tttgcggtga cgcaaagggt atggccagag atgtccatag atctttgcac 2040 acaattgctc aagaacaagg ttccatggat agtaccaaag ctgaaggttt cgtaaagaac 2100 ttacaaactt ccggtagata cttgagagat gtctggtga 2139
SEQ ID NO:!
A. thaliana
MSSSSSSSTS MIDLMAAIIK GEPVIVSDPA NASAYESVAA ELSSMLIENR QFAMIVTTSI 60 AVLIGCIVML VWRRSGSGNS KRVEPLKPLV IKPREEEIDD GRKKVTI FFG TQTGTAEGFA 120 KALGEEAKAR YEKTRFKIVD LDDYAADDDE YEEKLKKEDV AFFFLATYGD GEPTDNAARF 180 YKWFTEGNDR GEWLKNLKYG VFGLGNRQYE HFNKVAKVVD DILVEQGAQR LVQVGLGDDD 240 QCIEDDFTAW REALWPELDT ILREEGDTAV ATPYTAAVLE YRVSIHDSED AKFNDITLAN 300 GNGYTVFDAQ HPYKANVAVK RELHTPESDR SCIHLEFDIA GSGLTMKLGD HVGVLCDNLS 360 ETVDEALRLL DMSPDTYFSL HAEKEDGTPI SSSLPPPFPP CNLRTALTRY ACLLSSPKKS 420 ALVALAAHAS DPTEAERLKH LASPAGKDEY SKWWESQRS LLEVMAEFPS AKPPLGVFFA 480 GVAPRLQPRF YSISSSPKIA ETRIHVTCAL VYEKMPTGRI HKGVCSTWMK NAVPYEKSEK 540 LFLGRPIFVR QSNFKLPSDS KVPIIMIGPG TGLAPFRGFL QERLALVESG VELGPSVLFF 600 GCRNRRMDFI YEEELQRFVE SGALAELSVA FSREGPTKEY VQHKMMDKAS DIWNMISQGA 660 YLYVCGDAKG MARDVHRSLH TIAQEQGSMD STKAEGFVKN LQTSGRYLRD VW 712
SEQ ID NO:93
S. rebaudiana
atggaagcct cttacctata catttctatt ttgcttttac tggcatcata cctgttcacc 60 actcaactta gaaggaagag cgctaatcta ccaccaaccg tgtttccatc aataccaatc 120 attggacact tatacttact caaaaagcct ctttatagaa ctttagcaaa aattgccgct 180 aagtacggac caatactgca attacaactc ggctacagac gtgttctggt gatttcctca 240 ccatcagcag cagaagagtg ctttaccaat aacgatgtaa tcttcgcaaa tagacctaag 300 acattgtttg gcaaaatagt gggtggaaca tcccttggca gtttatccta cggcgatcaa 360 tggcgtaatc taaggagagt agcttctatc gaaatcctat cagttcatag gttgaacgaa 420 tttcatgata tcagagtgga tgagaacaga ttgttaatta gaaaacttag aagttcatct 480 tctcctgtta ctcttataac agtcttttat gctctaacat tgaacgtcat tatgagaatg 540 atctctggca aaagatattt cgacagtggg gatagagaat tggaggagga aggtaagaga 600 tttcgagaaa tcttagacga aacgttgctt ctagccggtg cttctaatgt tggcgactac 660 ttaccaatat tgaactggtt gggagttaag tctcttgaaa agaaattgat cgctttgcag 720 aaaaagagag atgacttttt ccagggtttg attgaacagg ttagaaaatc tcgtggtgct 780 aaagtaggca aaggtagaaa aacgatgatc gaactcttat tatctttgca agagtcagaa 840 cctgagtact atacagatgc tatgataaga tcttttgtcc taggtctgct ggctgcaggt 900 agtgatactt cagcgggcac tatggaatgg gccatgagct tactggtcaa tcacccacat 960 gtattgaaga aagctcaagc tgaaatcgat agagttatcg gtaataacag attgattgac 1020 gagtcagaca ttggaaatat cccttacatc gggtgtatta tcaatgaaac tctaagactc 1080 tatccagcag ggccattgtt gttcccacat gaaagttctg ccgactgcgt tatttccggt 1140 tacaatatac ctagaggtac aatgttaatc gtaaaccaat gggcgattca tcacgatcct 1200 aaagtctggg atgatcctga aacctttaaa cctgaaagat ttcaaggatt agaaggaact 1260 agagatggtt tcaaacttat gccattcggt tctgggagaa gaggatgtcc aggtgaaggt 1320 ttggcaataa ggctgttagg gatgacacta ggctcagtga tccaatgttt tgattgggag 1380 agagtaggag atgagatggt tgacatgaca gaaggtttgg gtgtcacact tcctaaggcc 1440 gttccattag ttgccaaatg taagccacgt tccgaaatga ctaatctcct atccgaactt 1500 taa 1503
SEQ ID NO:94
S. rebaudiana
MEASYLYISI LLLLASYLFT TQLRRKSANL PPTVFPSIPI IGHLYLLKKP LYRTLAKIAA 60
KYGPILQLQL GYRRVLVISS PSAAEECFTN NDVIFANRPK TLFGKIVGGT SLGSLSYGDQ 120
WRNLRRVASI EILSVHRLNE FHDIRVDENR LLIRKLRSSS SPVTLITVFY ALTLNVIMRM 180
ISGKRYFDSG DRELEEEGKR FREILDETLL LAGASNVGDY LPILNWLGVK SLEKKLIALQ 240
KKRDDFFQGL IEQVRKSRGA KVGKGRKTMI ELLLSLQESE PEYYTDAMIR SFVLGLLAAG 300
SDTSAGTMEW AMSLLVNHPH VLKKAQAEID RVIGNNRLID ESDIGNIPYI GCIINETLRL 360
YPAGPLLFPH ESSADCVISG YNIPRGTMLI VNQWAIHHDP KVWDDPETFK PERFQGLEGT 420
RDGFKLMPFG SGRRGCPGEG LAIRLLGMTL GSVIQCFDWE RVGDEMVDMT EGLGVTLPKA 480
VPLVAKCKPR SEMTNLLSEL 500
SEQ ID NO:95
atggaagtaa cagtagctag tagtgtagcc ctgagcctgg tctttattag catagtagta 60 agatgggcat ggagtgtggt gaattgggtg tggtttaagc cgaagaagct ggaaagattt 120 ttgagggagc aaggccttaa aggcaattcc tacaggtttt tatatggaga catgaaggag 180 aactctatcc tgctcaaaca agcaagatcc aaacccatga acctctccac ctcccatgac 240 atagcacctc aagtcacccc ttttgtcgac caaaccgtga aagcttacgg taagaactct 300 tttaattggg ttggccccat accaagggtg aacataatga atccagaaga tttgaaggac 360 gtcttaacaa aaaatgttga ctttgttaag ccaatatcaa acccacttat caagttgcta 420 gctacaggta ttgcaatcta tgaaggtgag aaatggacta aacacagaag gattatcaac 480 ccaacattcc attcggagag gctaaagcgt atgttacctt catttcacca aagttgtaat 540 gagatggtca aggaatggga gagcttggtg tcaaaagagg gttcatcatg tgagttggat 600 gtctggcctt ttcttgaaaa tatgtcggca gatgtgatct cgagaacagc atttggaact 660 agctacaaaa aaggacagaa aatctttgaa ctcttgagag agcaagtaat atatgtaacg 720 aaaggctttc aaagttttta cattccagga tggaggtttc tcccaactaa gatgaacaag 780 aggatgaatg agattaacga agaaataaaa ggattaatca ggggtattat aattgacaga 840 gagcaaatca ttaaggcagg tgaagaaacc aacgatgact tattaggtgc acttatggag 900 tcaaacttga aggacattcg ggaacatggg aaaaacaaca aaaatgttgg gatgagtatt 960 gaagatgtaa ttcaggagtg taagctgttt tactttgctg ggcaagaaac cacttcagtg 1020 ttgctggctt ggacaatggt tttacttggt caaaatcaga actggcaaga tcgagcaaga 1080 caagaggttt tgcaagtctt tggaagcagc aagccagatt ttgatggtct agctcacctt 1140 aaagtcgtaa ccatgatttt gcttgaagtt cttcgattat acccaccagt cattgaactt 1200 attcgaacca ttcacaagaa aacacaactt gggaagctct cactaccaga aggagttgaa 1260 gtccgcttac caacactgct cattcaccat gacaaggaac tgtggggtga tgatgcaaac 1320 cagttcaatc cagagaggtt ttcggaagga gtttccaaag caacaaagaa ccgactctca 1380 ttcttcccct tcggagccgg tccacgcatt tgcattggac agaacttttc tatgatggaa 1440 gcaaagttgg ccttagcatt gatcttgcaa cacttcacct ttgagctttc tccatctcat 1500 gcacatgctc cttcccatcg tataaccctt caaccacagt atggtgttcg tatcatttta 1560 catcgacgtt ag 1572
SEQ ID NO:96
R. suavissimus
atggaagtca ctgtcgcctc ttctgtcgct ttatccttag tcttcatttc cattgtcgtc 60 agatgggctt ggtccgttgt caactgggtt tggttcaaac caaagaagtt ggaaagattc 120 ttgagagagc aaggtttgaa gggtaattct tatagattct tgtacggtga catgaaggaa 180 aattctattt tgttgaagca agccagatcc aaaccaatga acttgtctac ctctcatgat 240 attgctccac aagttactcc attcgtcgat caaactgtta aagcctacgg taagaactct 300 ttcaattggg ttggtccaat tcctagagtt aacatcatga acccagaaga tttgaaggat 360 gtcttgacca agaacgttga cttcgttaag ccaatttcca acccattgat taaattgttg 420 gctactggta ttgccattta cgaaggtgaa aagtggacta agcatagaag aatcatcaac 480 cctaccttcc actctgaaag attgaagaga atgttaccat ctttccatca atcctgtaat 540 gaaatggtta aggaatggga atccttggtt tctaaagaag gttcttcttg cgaattggat 600 gtttggccat tcttggaaaa tatgtctgct gatgtcattt ccagaaccgc tttcggtacc 660 tcctacaaga agggtcaaaa gattttcgaa ttgttgagag agcaagttat ttacgttacc 720 aagggtttcc aatccttcta catcccaggt tggagattct tgccaactaa aatgaacaag 780 cgtatgaacg agatcaacga agaaattaaa ggtttgatca gaggtattat tatcgacaga 840 gaacaaatta ttaaagctgg tgaagaaacc aacgatgatt tgttgggtgc tttgatggag 900 tccaacttga aggatattag agaacatggt aagaacaaca agaatgttgg tatgtctatt 960 gaagatgtta ttcaagaatg taagttattc tacttcgctg gtcaagagac cacttctgtt 1020 ttgttagcct ggactatggt cttgttaggt caaaaccaaa attggcaaga tagagctaga 1080 caagaagttt tgcaagtctt cggttcttcc aagccagact ttgatggttt ggcccacttg 1140 aaggttgtta ctatgatttt gttagaagtt ttgagattgt acccaccagt cattgagtta 1200 atcagaacca ttcataaaaa gactcaattg ggtaaattat ctttgccaga aggtgttgaa 1260 gtcagattac caaccttgtt gattcaccac gataaggaat tatggggtga cgacgctaat 1320 caatttaatc cagaaagatt ttccgaaggt gtttccaagg ctaccaaaaa ccgtttgtcc 1380 ttcttcccat ttggtgctgg tccacgtatt tgtatcggtc aaaacttttc catgatggaa 1440 gccaagttgg ctttggcttt aatcttgcaa cacttcactt tcgaattgtc tccatcccat 1500 gcccacgctc cttctcatag aatcacttta caaccacaat acggtgtcag aatcatctta 1560 cacagaagat aa 1572
SEQ ID NO:97
R. suavissimus
MEVTVASSVA LSLVFISIVV RWAWSWNWV WFKPKKLERF LREQGLKGNS YRFLYGDMKE 60
NSILLKQARS KPMNLSTSHD IAPQVTPFVD QTVKAYGKNS FNWVGPIPRV NIMNPEDLKD 120
VLTKNVDFVK PISNPLIKLL ATGIAIYEGE KWTKHRRI IN PTFHSERLKR MLPSFHQSCN 180
EMVKEWESLV SKEGSSCELD VWPFLENMSA DVISRTAFGT SYKKGQKIFE LLREQVIYVT 240
KGFQSFYIPG WRFLPTKMNK RMNEINEEIK GLIRGIIIDR EQIIKAGEET NDDLLGALME 300
SNLKDIREHG KNNKNVGMSI EDVIQECKLF YFAGQETTSV LLAWTMVLLG QNQNWQDRAR 360
QEVLQVFGSS KPDFDGLAHL KWTMILLEV LRLYPPVIEL IRTIHKKTQL GKLSLPEGVE 420
VRLPTLLIHH DKELWGDDAN QFNPERFSEG VSKATKNRLS FFPFGAGPRI CIGQNFSMME 480
AKLALALILQ HFTFELSPSH AHAPSHRITL QPQYGVRIIL HRR 523
SEQ ID NO:98
atggaagcat caagggctag ttgtgttgcg ctatgtgttg tttgggtgag catagtaatt 60 acattggcat ggagggtgct gaattgggtg tggttgaggc caaagaaact agaaagatgc 120 ttgagggagc aaggccttac aggcaattct tacaggcttt tgtttggaga caccaaggat 180 ctctcgaaga tgctggaaca aacacaatcc aaacccatca aactctccac ctcccatgat 240 atagcgccac gagtcacccc atttttccat cgaactgtga actctaatgg caagaattct 300 tttgtttgga tgggccctat accaagagtg cacatcatga atccagaaga tttgaaagat 360 gccttcaaca gacatgatga ttttcataag acagtaaaaa atcctatcat gaagtctcca 420 ccaccgggca ttgtaggcat tgaaggtgag caatgggcta aacacagaaa gattatcaac 480 ccagcattcc atttagagaa gctaaagggt atggtaccaa tattttacca aagttgtagc 540 gagatgatta acaaatggga gagcttggtg tccaaagaga gttcatgtga gttggatgtg 600 tggccttatc ttgaaaattt taccagcgat gtgatttccc gagctgcatt tggaagtagc 660 tatgaagagg gaaggaaaat atttcaacta ctaagagagg aagcaaaagt ttattcggta 720 gctctacgaa gtgtttacat tccaggatgg aggtttctac caaccaagca gaacaagaag 780 acgaaggaaa ttcacaatga aattaaaggc ttacttaagg gcattataaa taaaagggaa 840 gaggcgatga aggcagggga agccactaaa gatgacttac taggaatact tatggagtcc 900 aacttcaggg aaattcagga acatgggaac aacaaaaatg ctggaatgag tattgaagat 960 gtaattggag agtgtaagtt gttttacttt gctgggcaag agaccacttc ggtgttgctt 1020 gtttggacaa tgattttact aagccaaaat caggattggc aagctcgtgc aagagaagag 1080 gtcttgaaag tctttggaag caacatccca acctatgaag agctaagtca cctaaaagtt 1140 gtgaccatga ttttacttga agttcttcga ttatacccat cagtcgttgc gcttcctcga 1200 accactcaca agaaaacaca gcttggaaaa ttatcattac cagctggagt ggaagtctcc 1260 ttgcccatac tgcttgttca ccatgacaaa gagttgtggg gtgaggatgc aaatgagttc 1320 aagccagaga ggttttcaga gggagtttca aaggcaacaa agaacaaatt tacatactta 1380 cctttcggag ggggtccaag gatttgcatt ggacaaaact ttgccatggt ggaagctaaa 1440 ttggccttgg ccctgatttt acaacacttt gcctttgagc tttctccatc ctatgctcat 1500 gctccttctg cagttataac ccttcaacct caatttggtg ctcatatcat tttgcataaa 1560 cgttga 1566
SEQ ID NO:99
atggaagctt ctagagcatc ttgtgttgct ttgtgtgttg tttgggtttc catcgttatt 60 actttggctt ggagagtttt gaattgggtc tggttaagac caaaaaagtt ggaaagatgc 120 ttgagagaac aaggtttgac tggtaactct tacagattgt tgttcggtga taccaaggac 180 ttgtctaaga tgttggaaca aactcaatcc aagcctatca agttgtctac ctctcatgat 240 attgctccaa gagttactcc attcttccat agaactgtta actccaacgg taagaactct 300 tttgtttgga tgggtccaat tccaagagtc catattatga accctgaaga tttgaaggac 360 gctttcaaca gacatgatga tttccataag accgtcaaga acccaattat gaagtctcca 420 ccaccaggta tagttggtat tgaaggtgaa caatgggcca aacatagaaa gattattaac 480 ccagccttcc acttggaaaa gttgaaaggt atggttccaa tcttctacca atcctgctct 540 gaaatgatta acaagtggga atccttggtt tccaaagaat cttcctgtga attggatgtc 600 tggccatatt tggaaaactt cacctccgat gttatttcca gagctgcttt tggttcttct 660 tacgaagaag gtagaaagat cttccaatta ttgagagaag aagccaaggt ttactccgtt 720 gctttgagat ctgtttacat tccaggttgg agattcttgc caactaagca aaacaaaaag 780 accaaagaaa tccacaacga aatcaagggt ttgttgaagg gtatcatcaa caagagagaa 840 gaagctatga aggctggtga agctacaaaa gatgatttgt tgggtatctt gatggaatcc 900 aacttcagag aaatccaaga acacggtaac aacaagaatg ccggtatgtc tattgaagat 960 gttatcggtg aatgcaagtt gttctacttt gctggtcaag aaactacctc cgttttgttg 1020 gtttggacca tgattttgtt gtcccaaaat caagattggc aagctagagc tagagaagaa 1080 gtcttgaaag ttttcggttc taacatccca acctacgaag aattgtctca cttgaaggtt 1140 gtcactatga tcttgttgga agtattgaga ttatacccat ccgttgttgc attgccaaga 1200 actactcata agaaaactca attgggtaaa ttgtccttgc cagctggtgt tgaagtttct 1260 ttgccaattt tgttagtcca ccacgacaaa gaattgtggg gtgaagatgc taatgaattc 1320 aagccagaaa gattctccga aggtgtttct aaagctacca agaacaagtt cacttacttg 1380 ccatttggtg gtggtccaag aatatgtatt ggtcaaaatt tcgctatggt cgaagctaaa 1440 ttggctttgg ctttgatctt gcaacatttc gctttcgaat tgtcaccatc ttatgctcat 1500 gctccatctg ctgttattac attgcaacca caatttggtg cccatatcat cttgcataag 1560 agataac 1567
SEQ ID NO:100
Prunus avium
MEASRASCVA LCVVWVSIVI TLAWRVLNWV WLRPKKLERC LREQGLTGNS YRLLFGDTKD 60
LSKMLEQTQS KPIKLSTSHD IAPRVTPFFH RTVNSNGKNS FVWMGPI PRV HIMNPEDLKD 120
AFNRHDDFHK TVKNPIMKSP PPGIVGIEGE QWAKHRKI IN PAFHLEKLKG MVPIFYQSCS 180
EMINKWESLV SKESSCELDV WPYLENFTSD VISRAAFGSS YEEGRKI FQL LREEAKVYSV 240
ALRSVYIPGW RFLPTKQNKK TKEIHNEIKG LLKGIINKRE EAMKAGEATK DDLLGILMES 300
NFREIQEHGN NKNAGMSIED VIGECKLFYF AGQETTSVLL WTMILLSQN QDWQARAREE 360
VLKVFGSNIP TYEELSHLKV VTMILLEVLR LYPSVVALPR TTHKKTQLGK LSLPAGVEVS 420
LPILLVHHDK ELWGEDANEF KPERFSEGVS KATKNKFTYL PFGGGPRICI GQNFAMVEAK 480
LALALILQHF AFELSPSYAH APSAVITLQP QFGAHIILHK R 521
SEQ ID NO:101
Prunus mume ASWVAVLSW WVSMVIAWAW RVLNWVWLRP KKLEKCLREQ GLAGNSYRLL FGDTKDLSKM 60
LEQTQSKPIK LSTSHDIAPH VTPFFHQTVN SYGKNSFVWM GPIPRVHIMN PEDLKDTFNR 120
HDDFHKWKN PIMKSLPQGI VGIEGEQWAK HRKIINPAFH LEKLKGMVPI FYRSCSEMIN 180
KWESLVSKES SCELDVWPYL ENFTSDVISR AAFGSSYEEG RKIFQLLREE AKIYTVAMRS 240
VYIPGWRFLP TKQNKKAKEI HNEIKGLLKG IINKREEAMK AGEATKDDLL GILMESNFRE 300
IQEHGNNKNA GMSIEDVIGE CKLFYFAGQE TTSVLL TM VLLSQNQDWQ ARAREEVLQV 360
FGSNI PTYEE LSQLKVVTMI LLEVLRLYPS WALPRTTHK KTQLGKLSLP AGVEVSLPIL 420
LVHHDKELWG EDANEFKPER FSEGVSKATK NQFTYFPFGG GPRICIGQNF AMMEAKLALS 480
LILRHFALEL SPLYAHAPSV TITLQPQYGA HIILHKR 517
SEQ ID NO:102
Prunus mume
MEASRPSCVA LSVVLVSIVI AWAWRVLNWV WLRPNKLERC LREQGLTGNS YRLLFGDTKE 60 ISMMVEQAQS KPIKLSTTHD IAPRVIPFSH QIVYTYGRNS FVWMGPTPRV TIMNPEDLKD 120 AFNKSDEFQR AISNPIVKSI SQGLSSLEGE KWAKHRKI IN PAFHLEKLKG MLPTFYQSCS 180 EMINKWESLV FKEGSREMDV WPYLENLTSD VISRAAFGSS YEEGRKI FQL LREEAKFYTI 240 AARSVYIPGW RFLPTKQNKR MKEIHKEVRG LLKGI INKRE DAIKAGEAAK GNLLGILMES 300 NFREIQEHGN NKNAGMSIED VIGECKLFYF AGQETTSVLL VWTLVLLSQN QDWQARAREE 360 VLQVFGTNIP TYDQLSHLKV VTMILLEVLR LYPAVVELPR TTYKKTQLGK FLLPAGVEVS 420 LHIMLAHHDK ELWGEDAKEF KPERFSEGVS KATKNQFTYF PFGAGPRICI GQNFAMLEAK 480 LALSLILQHF TFELSPSYAH APSVTITLHP QFGAHFILHK R 521
SEQ ID NO:103
Prunus mume
CVALSWLVS IVIAWAWRVL NWVWLRPNKL ERCLREQGLT GNSYRLLFGD TKEISMMVEQ 60 AQSKPIKLST THDIAPRVIP FSHQIVYTYG RNSFVWMGPT PRVTIMNPED LKDAFNKSDE 120 FQRAISNPIV KSISQGLSSL EGEKWAKHRK IINPAFHLEK LKGMLPTFYQ SCSEMINKWE 180 SLVFKEGSRE MDVWPYLENL TSDVISRAAF GSSYEEGRKI FQLLREEAKF YTIAARSVYI 240 PGWRFLPTKQ NKRMKEIHKE VRGLLKGIIN KREDAIKAGE AAKGNLLGIL MESNFREIQE 300 HGNNKNAGMS IEDVIGECKL FYFAGQETTS VLLVWTLVLL SQNQDWQARA REEVLQVFGT 360 NI PTYDQLSH LKVVTMILLE VLRLYPAWE LPRTTYKKTQ LGKFLLPAGV EVSLHIMLAH 420 HDKELWGEDA KEFKPERFSE GVSKATKNQF TYFPFGAGPR ICIGQNFAML EAKLALSLIL 480 QHFTFELSPS YAHAPSVTIT LHPQFGAHFI LHKR 514
SEQ ID NO:104
Prunus persica
MGPIPRVHIM NPEDLKDTFN RHDDFHKWK NPIMKSLPQG IVGIEGDQWA KHRKIINPAF 60
HLEKLKGMVP IFYQSCSEMI NIWKSLVSKE SSCELDVWPY LENFTSDVIS RAAFGSSYEE 120
GRKIFQLLRE EAKVYTVAVR SVYIPGWRFL PTKQNKKTKE IHNEIKGLLK GIINKREEAM 180
KAGEATKDDL LGILMESNFR EIQEHGNNKN AGMSIEDVIG ECKLFYFAGQ ETTSVLLVWT 240
MVLLSQNQDW QARAREEVLQ VFGSNIPTYE ELSHLKWTM ILLEVLRLYP SVVALPRTTH 300
KKTQLGKLSL PAGVEVSLPI LLVHHDKELW GEDANEFKPE RFSEGVSKAT KNQFTYFPFG 360
GGPRICIGQN FAMMEAKLAL SLILQHFTFE LSPQYSHAPS VTITLQPQYG AHLILHKR 418
SEQ ID NO:105
atgggtttgt tcccattaga ggattcctac gcgctggtct ttgaaggact agcaataaca 60 ctggctttgt actatctact gtctttcatc tacaaaacat ctaaaaagac atgtacacct 120 cctaaagcat ctggtgaaat cattccaatt acaggaatca tattgaatct gctatctggc 180 tcaagtggtc tacctattat cttagcactt gcctctttag cagacagatg tggtcctatt 240 ttcaccatta ggctgggtat taggagagtg ctagtagtat caaattggga aatcgctaag 300 gagattttca ctacccacga tttgatagtt tctaatagac caaaatactt agccgctaag 360 attcttggtt tcaattatgt ttcattctct ttcgctccat acggcccata ttgggtcgga 420 atcagaaaga ttattgctac aaaactaatg tcttcttcca gacttcagaa gttgcaattt 480 gtaagagttt ttgaactaga aaactctatg aaatctatca gagaatcatg gaaggagaaa 540 aaggatgaag agggaaaggt attagttgag atgaaaaagt ggttctggga actgaatatg 600 aacatagtgt taaggacagt tgctggtaaa caatacactg gtacagttga tgatgccgat 660 gcaaagcgta tctccgagtt attcagagaa tggtttcact acactggcag atttgtcgtt 720 ggagacgctt ttccttttct aggttggttg gacctgggcg gatacaaaaa gacaatggaa 780 ttagttgcta gtagattgga ctcaatggtc agtaaatggt tagatgagca tcgtaaaaag 840 caagctaacg atgacaaaaa ggaggatatg gatttcatgg atatcatgat ctccatgaca 900 gaagcaaatt caccacttga aggatacggc actgatacta ttatcaagac cacatgtatg 960 actttgattg tttcaggagt tgatacaacc tcaatcgtac ttacttgggc cttatcactt 1020 ttgttaaaca acagagatac tttgaaaaag gcacaagagg aattagatat gtgcgtaggt 1080 aaaggaagac aagtcaacga gtctgatctt gttaacttga tatacttgga agcagtgctt 1140 aaagaggctt taagacttta cccagcagcg ttcttaggcg gaccaagagc attcttggaa 1200 gattgtactg ttgctggtta tagaattcca aagggcacct gcttgttgat taacatgtgg 1260 aaactgcata gagatccaaa catttggagt gatccttgcg aattcaagcc agaaagattt 1320 ttgacaccta atcaaaagga tgttgatgtg atcggtatgg atttcgaatt gataccattt 1380 ggtgccggca gaagatattg tccaggtact agattggctt tacagatgtt gcatatcgta 1440 ttagcgacat tgctgcaaaa cttcgaaatg tcaacaccaa acgatgcgcc agtcgatatg 1500 actgcttctg ttggcatgac aaatgccaaa gcatcacctt tagaagtctt gctatcacct 1560 cgtgttaaat ggtcctaa 1578
SEQ ID NO:106
Stevia rebaudiana
MGLFPLEDSY ALVFEGLAIT LALYYLLSFI YKTSKKTCTP PKASGEHPIT GHLNLLSGSS 60
GLPHLALASL ADRCGPIFTI RLGIRRVLW SNWEIAKEIF TTHDLIVSNR PKYLAAKILG 120
FNYVSFSFAP YGPYWVGIRK IIATKLMSSS RLQKLQFVRV FELENSMKSI RESWKEKKDE 180
EGKVLVEMKK WFWELNMNIV LRTVAGKQYT GTVDDADAKR ISELFREWFH YTGRFWGDA 240
FPFLGWLDLG GYKKTMELVA SRLDSMVSKW LDEHRKKQAN DDKKEDMDFM DIMISMTEAN 300
SPLEGYGTDT IIKTTCMTLI VSGVDTTSIV LTWALSLLLN NRDTLKKAQE ELDMCVGKGR 360
QVNESDLVNL IYLEAVLKEA LRLYPAAFLG GPRAFLEDCT VAGYRI PKGT CLLINMWKLH 420
RDPNIWSDPC EFKPERFLTP NQKDVDVIGM DFELIPFGAG RRYCPGTRLA LQMLHIVLAT 480
LLQNFEMSTP NDAPVDMTAS VGMTNAKASP LEVLLSPRVK WS 522
SEQ ID NO:107
atgatacaag ttttaactcc aattctactc ttcctcatct tcttcgtttt ctggaaagtc 60 tacaaacatc aaaagactaa aatcaatcta ccaccaggtt ccttcggctg gccatttttg 120 ggtgaaacct tagccttact tagagcaggc tgggattctg agccagaaag attcgtaaga 180 gagcgtatca aaaagcatgg atctccactt gttttcaaga catcactatt tggagacaga 240 ttcgctgttc tttgcggtcc agctggtaat aagtttttgt tctgcaacga aaacaaatta 300 gtggcatctt ggtggccagt ccctgtaagg aagttgttcg gtaaaagttt actcacaata 360 agaggagatg aagcaaaatg gatgagaaaa atgctattgt cttacttggg tccagatgca 420 tttgccacac attatgccgt tactatggat gttgtaacac gtagacatat tgatgtccat 480 tggaggggca aggaggaagt taatgtattt caaacagtta agttgtacgc attcgaatta 540 gcttgtagat tattcatgaa cctagatgac ccaaaccaca tcgcgaaact cggtagtctt 600 ttcaacattt tcctcaaagg gatcatcgag cttcctatag acgttcctgg aactagattt 660 tactccagta aaaaggccgc agctgccatt agaattgaat tgaaaaagct cattaaagct 720 agaaaactcg aattgaagga gggtaaggcg tcttcttcac aggacttgct ttctcatcta 780 ttaacatcac ctgatgagaa tgggatgttc ttgacagaag aggaaatagt cgataacatt 840 ctacttttgt tattcgctgg tcacgatacc tctgcactat caataacact tttgatgaaa 900 accttaggtg aacacagtga tgtgtacgac aaggttttga aggaacaatt agaaatttcc 960 aaaacaaagg aggcttggga atcactaaag tgggaagata tccagaagat gaagtactca 1020 tggtcagtaa tctgtgaagt catgagattg aatcctcctg tcatagggac atacagagag 1080 gcgttggttg atatcgacta tgctggttac actatcccaa aaggatggaa gttgcattgg 1140 tcagctgttt ctactcaaag agacgaagcc aatttcgaag atgtaactag attcgatcca 1200 tccagatttg aaggggcagg ccctactcca ttcacatttg tgcctttcgg tggaggtcct 1260 agaatgtgtt taggcaaaga gtttgccagg ttagaagtgt tagcatttct ccacaacatt 1320 gttaccaact ttaagtggga tcttctaatc cctgatgaga agatcgaata tgatccaatg 1380 gctactccag ctaagggctt gccaattaga cttcatccac accaagtcta a 1431 SEQ ID NO:108
Stevia rebaudiana
MIQVLTPILL FLIFFVFWKV YKHQKTKINL PPGSFGWPFL GETLALLRAG WDSEPERFVR 60
ERIKKHGSPL VFKTSLFGDR FAVLCGPAGN KFLFCNENKL VASWWPVPVR KLFGKSLLTI 120
RGDEAKWMRK MLLSYLGPDA FATHYAVTMD WTRRHIDVH WRGKEEVNVF QTVKLYAFEL 180
ACRLFMNLDD PNHIAKLGSL FNIFLKGIIE LPIDVPGTRF YSSKKAAAAI RIELKKLIKA 240
RKLELKEGKA SSSQDLLSHL LTSPDENGMF LTEEEIVDNI LLLLFAGHDT SALSITLLMK 300
TLGEHSDVYD KVLKEQLEIS KTKEAWESLK WEDIQKMKYS WSVICEVMRL NPPVIGTYRE 360
ALVDIDYAGY TIPKGWKLHW SAVSTQRDEA NFEDVTRFDP SRFEGAGPTP FTFVPFGGGP 420
RMCLGKEFAR LEVLAFLHNI VTNFKWDLLI PDEKIEYDPM ATPAKGLPIR LHPHQV 476
SEQ ID NO:109
atggagtctt tagtggttca tacagtaaat gctatctggt gtattgtaat cgtcgggatt 60 ttctcagttg gttatcacgt ttacggtaga gctgtggtcg aacaatggag aatgagaaga 120 tcactgaagc tacaaggtgt taaaggccca ccaccatcca tcttcaatgg taacgtctca 180 gaaatgcaac gtatccaatc cgaagctaaa cactgctctg gcgataacat tatctcacat 240 gattattctt cttcattatt cccacacttc gatcactgga gaaaacagta cggcagaatc 300 tacacatact ctactggatt aaagcaacac ttgtacatca atcatccaga aatggtgaag 360 gagctatctc agactaacac attgaacttg ggtagaatca cccatataac caaaagattg 420 aatcctatct taggtaacgg aatcataacc tctaatggtc ctcattgggc ccatcagcgt 480 agaattatcg cctacgagtt tactcatgat aagatcaagg gtatggttgg tttgatggtt 540 gagtctgcta tgcctatgtt gaataagtgg gaggagatgg taaagagagg cggagaaatg 600 ggatgcgaca taagagttga tgaggacttg aaagatgttt cagcagatgt gattgcaaaa 660 gcctgtttcg gatcctcatt ttctaaaggt aaggctattt tctctatgat aagagatttg 720 cttacagcta tcacaaagag aagtgttcta ttcagattca acggattcac tgatatggtc 780 tttgggagta aaaagcatgg tgacgttgat atagacgctt tagaaatgga attggaatca 840 tccatttggg aaactgtcaa ggaacgtgaa atagaatgta aagatactca caaaaaggat 900 ctgatgcaat tgattttgga aggggcaatg cgttcatgtg acggtaacct ttgggataaa 960 tcagcatata gaagatttgt tgtagataat tgtaaatcta tctacttcgc agggcatgat 1020 agtacagctg tctcagtgtc atggtgtttg atgttactgg ccctaaaccc atcatggcaa 1080 gttaagatcc gtgatgaaat tctgtcttct tgcaaaaatg gtattccaga tgccgaaagt 1140 atcccaaacc ttaaaacagt gactatggtt attcaagaga caatgagatt ataccctcca 1200 gcaccaatcg tcgggagaga agcctctaaa gatatcagat tgggcgatct agttgttcct 1260 aaaggcgtct gtatatggac actaatacca gctttacaca gagatcctga gatttgggga 1320 ccagatgcaa acgatttcaa accagaaaga ttttctgaag gaatttcaaa ggcttgtaag 1380 tatcctcaaa gttacattcc atttggtctg ggtcctagaa catgcgttgg taaaaacttt 1440 ggcatgatgg aagtaaaggt tcttgtttcc ctgattgtct ccaagttctc tttcactcta 1500 tctcctacct accaacatag tcctagtcac aaacttttag tagaaccaca acatggggtg 1560 gtaattagag tggtttaa 1578
SEQ ID NO:1 10
Arabidopsis thaliana
MESLVVHTVN AIWCIVIVGI FSVGYHVYGR AWEQWRMRR SLKLQGVKGP PPSIFNGNVS 60
EMQRIQSEAK HCSGDNIISH DYSSSLFPHF DHWRKQYGRI YTYSTGLKQH LYINHPEMVK 120
ELSQTNTLNL GRITHITKRL NPILGNGIIT SNGPHWAHQR RIIAYEFTHD KIKGMVGLMV 180
ESAMPMLNKW EEMVKRGGEM GCDIRVDEDL KDVSADVIAK ACFGSSFSKG KAIFSMIRDL 240
LTAITKRSVL FRFNGFTDMV FGSKKHGDVD IDALEMELES SIWETVKERE IECKDTHKKD 300
LMQLILEGAM RSCDGNLWDK SAYRRFWDN CKSIYFAGHD STAVSVSWCL MLLALNPSWQ 360
VKIRDEILSS CKNGIPDAES I PNLKTVTMV IQETMRLYPP APIVGREASK DIRLGDLVVP 420
KGVCIWTLIP ALHRDPEIWG PDANDFKPER FSEGISKACK YPQSYIPFGL GPRTCVGKNF 480
GMMEVKVLVS LIVSKFSFTL SPTYQHSPSH KLLVEPQHGV VIRVV 525
SEQ ID NO:1 1 1
atgtacttcc tactacaata cctcaacatc acaaccgttg gtgtctttgc cacattgttt 60 ctctcttatt gtttacttct ctggagaagt agagcgggta acaaaaagat tgccccagaa 120 gctgccgctg catggcctat tatcggccac ctccacttac ttgcaggtgg atcccatcaa 180 ctaccacata ttacattggg taacatggca gataagtacg gtcctgtatt cacaatcaga 240 ataggcttgc atagagctgt agttgtctca tcttgggaaa tggcaaagga atgttcaaca 300 gctaatgatc aagtgtcttc ttcaagacct gaactattag cttctaagtt gttgggttat 360 aactacgcca tgtttggttt ttcaccatac ggttcatact ggagagaaat gagaaagatc 420 atctctctcg aattactatc taattccaga ttggaactat tgaaagatgt tagagcctca 480 gaagttgtca catctattaa ggaactatac aaattgtggg cggaaaagaa gaatgagtca 540 ggattggttt ctgtcgagat gaaacaatgg ttcggagatt tgactttaaa cgtgatcttg 600 agaatggtgg ctggtaaaag atacttctcc gcgagtgacg cttcagaaaa caaacaggcc 660 cagcgttgta gaagagtctt cagagaattc ttccatctct ccggcttgtt tgtggttgct 720 gatgctatac cttttcttgg atggctcgat tggggaagac acgagaagac cttgaaaaag 780 accgccatag aaatggattc catcgcccag gagtggcttg aggaacatag acgtagaaaa 840 gattctggag atgataattc tacccaagat ttcatggacg ttatgcaatc tgtgctagat 900 ggcaaaaatc taggcggata cgatgctgat acgattaaca aggctacatg cttaactctt 960 atatcaggtg gcagtgatac tactgtagtt tctttgacat gggctcttag tcttgtgtta 1020 aacaatagag atactttgaa aaaggcacag gaagagttag acatccaagt cggtaaggaa 1080 agattggtta acgagcaaga catcagtaag ttagtttact tgcaagcaat agtaaaagag 1140 acactcagac tttatccacc aggtcctttg ggtggtttga gacaattcac tgaagattgt 1200 acactaggtg gctatcacgt ttcaaaagga actagattaa tcatgaactt atccaagatt 1260 caaaaagatc cacgtatttg gtctgatcct actgaattcc aaccagagag attccttacg 1320 actcataaag atgtcgatcc acgtggtaaa cactttgaat tcattccatt cggtgcagga 1380 agacgtgcat gtcctggtat cacattcgga ttacaagtac tacatctaac attggcatct 1440 ttcttgcatg cgtttgaatt ttcaacacca tcaaatgagc aggttaacat gagagaatca 1500 ttaggtctta cgaatatgaa atctacccca ttagaagttt tgatttctcc aagactatcc 1560 cttaattgct tcaaccttat gaaaatttga 1590
SEQ ID NO:1 12
Vitis vinifera
MYFLLQYLNI TTVGVFATLF LSYCLLLWRS RAGNKKIAPE AAAAWPIIGH LHLLAGGSHQ 60
LPHITLGNMA DKYGPVFTIR IGLHRAVWS SWEMAKECST ANDQVSSSRP ELLASKLLGY 120
NYAMFGFSPY GSYWREMRKI ISLELLSNSR LELLKDVRAS EWTSIKELY KLWAEKKNES 180
GLVSVEMKQW FGDLTLNVIL RMVAGKRYFS ASDASENKQA QRCRRVFREF FHLSGLFVVA 240
DAIPFLGWLD WGRHEKTLKK TAIEMDSIAQ EWLEEHRRRK DSGDDNSTQD FMDVMQSVLD 300
GKNLGGYDAD TINKATCLTL ISGGSDTTW SLTWALSLVL NNRDTLKKAQ EELDIQVGKE 360
RLVNEQDISK LVYLQAIVKE TLRLYPPGPL GGLRQFTEDC TLGGYHVSKG TRLIMNLSKI 420
QKDPRIWSDP TEFQPERFLT THKDVDPRGK HFEFIPFGAG RRACPGITFG LQVLHLTLAS 480
FLHAFEFSTP SNEQVNMRES LGLTNMKSTP LEVLISPRLS SCSLYN 526
SEQ ID NO:1 13
atggaaccta acttttactt gtcattacta ttgttgttcg tgaccttcat ttctttaagt 60 ctgtttttca tcttttacaa acaaaagtcc ccattgaatt tgccaccagg gaaaatgggt 120 taccctatca taggtgaaag tttagaattc ctatccacag gctggaaggg acatcctgaa 180 aagttcatat ttgatagaat gcgtaagtac agtagtgagt tattcaagac ttctattgta 240 ggcgaatcca cagttgtttg ctgtggggca gctagtaaca aattcctatt ctctaacgaa 300 aacaaactgg taactgcctg gtggccagat tctgttaaca aaatcttccc aacaacttca 360 ctggattcta atttgaagga ggaatctata aagatgagaa agttgctgcc acagttcttc 420 aaaccagaag cacttcaaag atacgtcggc gttatggatg taatcgcaca aagacatttt 480 gtcactcact gggacaacaa aaatgagatc acagtttatc cacttgctaa aagatacact 540 ttcttgcttg cgtgtagact gttcatgtct gttgaggatg aaaatcatgt ggcgaaattc 600 tcagacccat tccaactaat cgctgcaggc atcatttcac ttcctatcga tcttcctggt 660 actccattca acaaggccat aaaggcttca aatttcatta gaaaagagct gataaagatt 720 atcaaacaaa gacgtgttga tctggcagag ggtacagcat ctccaaccca ggatatcttg 780 tcacatatgc tattaacatc tgatgaaaac ggtaaatcta tgaacgagtt gaacattgcc 840 gacaagattc ttggactatt gataggaggc cacgatacag cttcagtagc ttgcacattt 900 ctagtgaagt acttaggaga attaccacat atctacgata aagtctacca agagcaaatg 960 gaaattgcca agtccaaacc tgctggggaa ttgttgaatt gggatgactt gaaaaagatg 1020 aagtattcat ggaatgtggc atgtgaggta atgagattgt caccaccttt acaaggtggt 1080 tttagagagg ctataactga ctttatgttt aacggtttct ctattccaaa agggtggaag 1140 ttatactggt ccgccaactc tacacacaaa aatgcagaat gtttcccaat gcctgagaaa 1200 ttcgatccta ccagatttga aggtaatggt ccagcgcctt atacatttgt accattcggt 1260 ggaggcccta gaatgtgtcc tggaaaggaa tacgctagat tagaaatctt ggttttcatg 1320 cataatctgg tcaaacgttt taagtgggaa aaggttattc cagacgaaaa gattattgtc 1380 gatccattcc caatcccagc taaagatctt ccaatccgtt tgtatcctca caaagcttaa 1440
SEQ ID NO:1 14
Medicago truncatula
MEPNFYLSLL LLFVTFISLS LFFIFYKQKS PLNLPPGKMG YPIIGESLEF LSTGWKGHPE 60
KFIFDRMRKY SSELFKTSIV GESTWCCGA ASNKFLFSNE NKLVTAWWPD SVNKIFPTTS 120
LDSNLKEESI KMRKLLPQFF KPEALQRYVG VMDVIAQRHF VTHWDNKNEI TVYPLAKRYT 180
FLLACRLFMS VEDENHVAKF SDPFQLIAAG IISLPIDLPG TPFNKAIKAS NFIRKELIKI 240
IKQRRVDLAE GTASPTQDIL SHMLLTSDEN GKSMNELNIA DKILGLLIGG HDTASVACTF 300
LVKYLGELPH IYDKVYQEQM EIAKSKPAGE LLNWDDLKKM KYSWNVACEV MRLSPPLQGG 360
FREAITDFMF NGFSIPKGWK LYWSANSTHK NAECFPMPEK FDPTRFEGNG PAPYTFVPFG 420
GGPRMCPGKE YARLEILVFM HNLVKRFKWE KVIPDEKIIV DPFPIPAKDL PIRLYPHKA 479
SEQ ID NO:1 15
atggcctctg ttactttggg ttcctggatc gtcgtccacc accataacca tcaccatcca 60 tcatctatcc taactaaatc tcgttcaaga tcctgtccta ttacactaac caaaccaatc 120 tcttttcgtt caaagagaac agtttcctct agtagttcta tcgtgtcctc tagtgtcgtc 180 actaaggaag acaatctgag acagtctgaa ccttcttcct ttgatttcat gtcatatatc 240 attactaagg cagaactagt gaataaggct cttgattcag cagttccatt aagagagcca 300 ttgaaaatcc atgaagcaat gagatactct cttctagctg gcgggaagag agtcagacct 360 gtactctgca tagcagcgtg cgaattagtt ggtggcgagg aatcaaccgc tatgcctgcc 420 gcttgtgctg tagaaatgat tcatacaatg tcactgatac acgatgattt gccatgtatg 480 gataacgatg atctgagaag gggtaagcca actaaccata aggttttcgg cgaagatgtt 540 gccgtcttag ctggtgatgc tttgttatct ttcgcgttcg aacatttggc atccgcaaca 600 tcaagtgatg ttgtgtcacc agtaagagta gttagagcag ttggagaact ggctaaagct 660 attggaactg agggtttagt tgcaggtcaa gtcgtcgata tctcttccga aggtcttgat 720 ttgaatgatg taggtcttga acatctcgaa ttcatccatc ttcacaagac agctgcactt 780 ttagaagcca gtgcggttct cggcgcaatt gttggcggag ggagtgatga cgaaattgag 840 agattgagga agtttgctag atgtatagga ttactgttcc aagtagtaga cgatatacta 900 gatgtgacaa agtcttccaa agagttggga aaaacagctg gtaaagattt gattgccgac 960 aaattgacct accctaagat tatggggcta gaaaaatcaa gagaatttgc cgagaaactc 1020 aatagagagg cgcgtgatca actgttgggt ttcgattctg ataaagttgc accactctta 1080 gccttagcca actacatcgc ttacagacaa aactaa 1116
SEQ ID NO:1 16
Arabidopsis thaliana
MASVTLGSWI VVHHHNHHHP SSILTKSRSR SCPITLTKPI SFRSKRTVSS SSSIVSSSW 60
TKEDNLRQSE PSSFDFMSYI ITKAELVNKA LDSAVPLREP LKIHEAMRYS LLAGGKRVRP 120
VLCIAACELV GGEESTAMPA ACAVEMIHTM SLIHDDLPCM DNDDLRRGKP TNHKVFGEDV 180
AVLAGDALLS FAFEHLASAT SSDWSPVRV VRAVGELAKA IGTEGLVAGQ VVDISSEGLD 240
LNDVGLEHLE FIHLHKTAAL LEASAVLGAI VGGGSDDEIE RLRKFARCIG LLFQVVDDIL 300
DVTKSSKELG KTAGKDLIAD KLTYPKIMGL EKSREFAEKL NREARDQLLG FDSDKVAPLL 360
ALANYIAYRQ N 371
SEQ ID NO:1 17
R. suavissimus
MATLLEHFQA MPFAIPIALA ALSWLFLFYI KVSFFSNKSA QAKLPPVPW PGLPVIGNLL 60 QLKEKKPYQT FTRWAEEYGP IYSIRTGAST MVVLNTTQVA KEAMVTRYLS ISTRKLSNAL 120
KILTADKCMV AISDYNDFHK MIKRYILSNV LGPSAQKRHR SNRDTLRANV CSRLHSQVKN 180
SPREAVNFRR VFEWELFGIA LKQAFGKDIE KPIYVEELGT TLSRDEI FKV LVLDIMEGAI 240
EVDWRDFFPY LRWIPNTRME TKIQRLYFRR KAVMTALINE QKKRIASGEE INCYIDFLLK 300
EGKTLTMDQI SMLLWETVIE TADTTMVTTE WAMYEVAKDS KRQDRLYQEI QKVCGSEMVT 360
EEYLSQLPYL NAVFHETLRK HSPAALVPLR YAHEDTQLGG YYIPAGTEIA INIYGCNMDK 420
HQWESPEEWK PERFLDPKFD PMDLYKTMAF GAGKRVCAGS LQAMLIACPT IGRLVQEFEW 480
KLRDGEEENV DTVGLTTHKR YPMHAILKPR S 511
SEQ ID NO:1 18
atggctgaac aacaaaagat caagaagtct ccacacgttt tgttgattcc atttccattg 60 caaggtcaca tcaacccatt cattcaattc ggtaagagat tgatttccaa gggtgttaag 120 actactttgg ttactaccat ccataccttg aactctacct tgaaccattc taacactacc 180 accacctcca ttgaaattca agctatttcc gatggttgtg atgaaggtgg ttttatgtct 240 gctggtgaat cttacttgga aacctttaag caagttggtt ctaagtcctt ggccgatttg 300 attaagaagt tgcaatctga aggtactacc attgatgcca ttatctacga ttctatgacc 360 gaatgggttt tggatgttgc tattgaattc ggtattgatg gtggttcatt cttcactcaa 420 gcttgtgttg ttaactcctt gtactaccat gttcacaagg gtttgatctc attgccattg 480 ggtgaaactg tttctgttcc aggtttccca gttttacaaa gatgggaaac tccattgatc 540 ttgcaaaacc acgaacaaat tcaatctcca tggtcccaaa tgttgtttgg tcaattcgcc 600 aacattgatc aagctagatg ggtttttacc aactccttct acaagttgga agaagaagtt 660 atcgaatgga ccagaaagat ctggaacttg aaagttattg gtccaacctt gccatctatg 720 tacttggata agagattgga tgacgataag gacaacggtt tcaacttgta caaggctaac 780 catcatgaat gcatgaattg gttggacgac aagccaaaag aatccgttgt ttatgttgct 840 ttcggttctt tggtcaaaca tggtccagaa caagttgaag aaattaccag agccttgatc 900 gattccgatg ttaatttctt gtgggtcatc aagcacaaag aagaaggtaa attgccagaa 960 aacttgtccg aagttatcaa aactggtaag ggtttgattg tcgcttggtg taaacaattg 1020 gatgttttgg ctcatgaatc cgttggttgt ttcgttactc attgtggttt caactccacc 1080 ttggaagcta tttctttggg tgttccagtt gttgctatgc cacaattttc tgatcaaact 1140 accaacgcta agttgttgga cgaaattttg ggtgttggtg ttagagttaa ggctgacgaa 1200 aatggtatcg ttagaagagg taacttggct tcttgcatca agatgatcat ggaagaagaa 1260 agaggtgtca tcattagaaa gaacgctgtt aagtggaagg atttggctaa agttgctgtt 1320 catgaaggtg gtagttccga taatgatatc gttgaattcg tttccgaatt gatcaaggcc 1380 taa 1383
SEQ ID NO:1 19
gcacagcaca catcagaatc cgcagctgtc gcaaagggca gcagtttgac ccctatagtg 60 agaactgacg ctgagtcaag gagaacaaga tggccaaccg atgacgatga cgccgaacct 120 ttagtggatg agatcagggc aatgcttact tccatgtctg atggtgacat ttccgtgagc 180 gcatacgata cagcctgggt cggattggtt ccaagattag acggcggtga aggtcctcaa 240 tttccagcag ctgtgagatg gataagaaat aaccagttgc ctgacggaag ttggggcgat 300 gccgcattat tctctgccta tgacaggctt atcaataccc ttgcctgcgt tgtaactttg 360 acaaggtggt ccctagaacc agagatgaga ggtagaggac tatctttttt gggtaggaac 420 atgtggaaat tagcaactga agatgaagag tcaatgccta ttggcttcga attagcattt 480 ccatctttga tagagcttgc taagagccta ggtgtccatg acttccctta tgatcaccag 540 gccctacaag gaatctactc ttcaagagag atcaaaatga agaggattcc aaaagaagtg 600 atgcataccg ttccaacatc aatattgcac agtttggagg gtatgcctgg cctagattgg 660 gctaaactac ttaaactaca gagcagcgac ggaagttttt tgttctcacc agctgccact 720 gcatatgctt taatgaatac cggagatgac aggtgtttta gctacatcga tagaacagta 780 aagaaattca acggcggcgt ccctaatgtt tatccagtgg atctatttga acatatttgg 840 gccgttgata gacttgaaag attaggaatc tccaggtact tccaaaagga gatcgaacaa 900 tgcatggatt atgtaaacag gcattggact gaggacggta tttgttgggc aaggaactct 960 gatgtcaaag aggtggacga cacagctatg gcctttagac ttcttaggtt gcacggctac 1020 agcgtcagtc ctgatgtgtt taaaaacttc gaaaaggacg gtgaattttt cgcatttgtc 1080 ggacagtcta atcaagctgt taccggtatg tacaacttaa acagagcaag ccagatatcc 1140 ttcccaggcg aggatgtgct tcatagagct ggtgccttct catatgagtt cttgaggaga 1200 aaagaagcag agggagcttt gagggacaag tggatcattt ctaaagatct acctggtgaa 1260 gttgtgtata ctttggattt tccatggtac ggcaacttac ctagagtcga ggccagagac 1320 tacctagagc aatacggagg tggtgatgac gtttggattg gcaagacatt gtataggatg 1380 ccacttgtaa acaatgatgt atatttggaa ttggcaagaa tggatttcaa ccactgccag 1440 gctttgcatc agttagagtg gcaaggacta aaaagatggt atactgaaaa taggttgatg 1500 gactttggtg tcgcccaaga agatgccctt agagcttatt ttcttgcagc cgcatctgtt 1560 tacgagcctt gtagagctgc cgagaggctt gcatgggcta gagccgcaat actagctaac 1620 gccgtgagca cccacttaag aaatagccca tcattcagag aaaggttaga gcattctctt 1680 aggtgtagac ctagtgaaga gacagatggc tcctggttta actcctcaag tggctctgat 1740 gcagttttag taaaggctgt cttaagactt actgattcat tagccaggga agcacagcca 1800 atccatggag gtgacccaga agatattata cacaagttgt taagatctgc ttgggccgag 1860 tgggttaggg aaaaggcaga cgctgccgat agcgtgtgca atggtagttc tgcagtagaa 1920 caagagggat caagaatggt ccatgataaa cagacctgtc tattattggc tagaatgatc 1980 gaaatttctg ccggtagggc agctggtgaa gcagccagtg aggacggcga tagaagaata 2040 attcaattaa caggctccat ctgcgacagt cttaagcaaa aaatgctagt ttcacaggac 2100 cctgaaaaaa atgaagagat gatgtctcac gtggatgacg aattgaagtt gaggattaga 2160 gagttcgttc aatatttgct tagactaggt gaaaaaaaga ctggatctag cgaaaccagg 2220 caaacatttt taagtatagt gaaatcatgt tactatgctg ctcattgccc acctcatgtc 2280 gttgatagac acattagtag agtgattttc gagccagtaa gtgccgcaaa gtaaccgcgg 2340
SEQ ID NO:120
Zea mays
AQHTSESAAV AKGSSLTPIV RTDAESRRTR WPTDDDDAEP LVDEIRAMLT SMSDGDISVS 60
AYDTAWVGLV PRLDGGEGPQ FPAAVRWIRN NQLPDGSWGD AALFSAYDRL INTLACWTL 120
TRWSLEPEMR GRGLSFLGRN MWKLATEDEE SMPIGFELAF PSLIELAKSL GVHDFPYDHQ 180
ALQGIYSSRE IKMKRI PKEV MHTVPTSILH SLEGMPGLDW AKLLKLQSSD GSFLFSPAAT 240
AYALMNTGDD RCFSYIDRTV KKFNGGVPNV YPVDLFEHIW AVDRLERLGI SRYFQKEIEQ 300
CMDYVNRHWT EDGICWARNS DVKEVDDTAM AFRLLRLHGY SVSPDVFKNF EKDGEFFAFV 360
GQSNQAVTGM YNLNRASQIS FPGEDVLHRA GAFSYEFLRR KEAEGALRDK WIISKDLPGE 420
WYTLDFPWY GNLPRVEARD YLEQYGGGDD VWIGKTLYRM PLVNNDVYLE LARMDFNHCQ 480
ALHQLEWQGL KRWYTENRLM DFGVAQEDAL RAYFLAAASV YEPCRAAERL AWARAAILAN 540
AVSTHLRNSP SFRERLEHSL RCRPSEETDG SWFNSSSGSD AVLVKAVLRL TDSLAREAQP 600
IHGGDPEDII HKLLRSAWAE WVREKADAAD SVCNGSSAVE QEGSRMVHDK QTCLLLARMI 660
EISAGRAAGE AASEDGDRRI IQLTGSICDS LKQKMLVSQD PEKNEEMMSH VDDELKLRIR 720
EFVQYLLRLG EKKTGSSETR QTFLSIVKSC YYAAHCPPHV VDRHISRVIF EPVSAAK 777

Claims

WHAT IS CLAIMED IS:
1. A recombinant host cell capable of producing one or more steviol glycosides or glycosides of a steviol precursor, comprising a recombinant gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31, 49, 51, 67, 68, 73, 75, 79, 81, 83, 84, 86-88, 90, 91, 96, 99, 107, 110, 111, 113, 115, 119-121, 123, 128, 129, 135, 136, 140, 141, 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181, 183-189, 191-195, 200, 204, 209, 211, 212, 215, 221, 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301, 303, 310, 311, 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341, 346, 347, 357, 364, 370, 371, 373, 375-377, 380, 385, 387- 391, 396, 401, 407-411, 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4.
2. The recombinant host cell of claim 1, wherein the polypeptide comprises at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31S, G31A, T49I, N51K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91E, S96T, D99E, E107S, T110P, 1111V, A113C, 1115V, M119F, T120L, E121P, V123A, I128K, E129Q, G135A, S136A, Q140N, A141S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N211H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301N, D303N, 1310V, K311R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341E, E346P, E346K, S347A, F357W, I364L, V370M, V371I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391K, V396A, N401K, G407E, N408E, L409I, A410E, S411D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V. The recombinant host cell of claim 1 or 2, further comprising:
(a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);
(b) a gene encoding a polypeptide capable of synthesizing eni-copalyl diphosphate from GGPP;
(c) a gene encoding an a polypeptide capable of synthesizing eni-kaurene from en/-copalyl diphosphate;
(d) a gene encoding a polypeptide capable of synthesizing eni-kaurenoic acid from en/-kaurene;
(e) a gene encoding a polypeptide capable of synthesizing steviol from ent- kaurenoic acid;
(f) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;
(g) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;
(h) a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside; and/or
(i) a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside;
wherein at least one of the genes is a recombinant gene.
The recombinant host cell of claim 3, wherein:
(a) the polypeptide capable of synthesizing GGPP comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, or 1 16;
(b) the polypeptide capable of synthesizing enf-copalyl diphosphate comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:34, 36, 38, 40, or 42; (c) the polypeptide capable of synthesizing enf-kaurene comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:44, 46, 48, 50, or 52;
(d) the polypeptide capable of synthesizing en/-kaurenoic acid comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:60, 62, 66, 68, 70, 72, 74, 76, or 117;
(e) the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:78, 80, 82, 84, 86, 88, 90, or 92;
(f) the polypeptide capable of synthesizing steviol comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:94, 97, 100, 101 , 102, 103, 104, 106, 108, 1 10, 1 12, or 1 14;
(g) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(h) the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-0- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(i) the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.
5. The recombinant host cell of any one of claims 1-4, wherein the one or more steviol glycosides or glycosides of the steviol precursor comprises kaurenoate-19-O-glucoside (19-KMG), steviol-13-O-glucoside (13-SMG), steviol-1 ,2-Bioside, steviol-1 ,3-Bioside, steviol-19-O-glucoside (19-SMG), 1 ,2-Stevioside, 1 ,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (Rebl), dulcoside A, a mono-glycosylated ent- kaurenoic acid, a di-glycosylated enf-kaurenoic acid, a tri-glycosylated eni-kaurenoic acid, a mono-glycosylated enf-kaurenols, a di-glycosylated enf-kaurenol, a tri- glycosylated en/-kaurenol, a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and/or an isomer thereof.
The recombinant host cell of any of claims 1-5, wherein expression of the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having the one or more amino acid substitutions, increases or decreases the amount of 19-KMG, 19-SMG, and/or rubusoside produced by the cell by at least about 5%, 10%, 25%, 50%, or 100% relative to a corresponding host expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group lacking the one or more amino acid substitutions.
The recombinant host cell of any of claims 1 -6, wherein the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, an archaeal cell, or a bacterial cell.
The recombinant host cell of claim 1 , wherein the recombinant host cell is a Saccharomyces cerevisiae cell.
The recombinant host cell of claim 1 , wherein the recombinant host cell is a Yarrowia lipolytica cell.
A method of producing one or more steviol glycosides or glycosides of a steviol precursor in a cell culture, comprising culturing the recombinant host cell of any one of claims 1-9 in the cell culture, under conditions in which the genes are expressed; and wherein the one or more steviol glycosides or glycosides of the steviol precursor is produced by the recombinant host cell.
1 1. The method of claim 10, wherein the genes are constitutively expressed.
12. The method of claim 10, wherein the expression of the genes is induced.
13. The method of any one of claims 10-12, further comprising isolating the produced one or more steviol glycosides or glycosides of the steviol precursor from the cell culture.
14. The method of claim 13, wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or glycosides of the steviol precursor, and:
(a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or
(b) contacting the supernatant with one or more ion exchange or reversed- phase chromatography columns in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or
(c) crystallizing or extracting the produced one or more steviol glycosides or glycosides of the steviol precursor;
thereby isolating the produced one or more steviol glycosides or glycosides of the steviol precursor.
15. The method of any one of claims 10-12, further comprising recovering the one or more steviol glycosides or glycosides of the steviol precursor from the cell culture.
16. The method of claim 15, wherein the recovered one or more steviol glycosides or glycosides of the steviol precursor is enriched for the one or more steviol glycosides or glycosides of the steviol precursor relative to a steviol glycoside composition of Stevia plant and has a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract.
A method for producing one or more steviol glycosides or glycosides of a steviol precursor, comprising whole-cell bioconversion of a plant-derived or synthetic steviol, steviol precursors, and/or steviol glycosides in a cell culture of a recombinant host cell using a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 119-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191-195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-41 1 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4;
and, optionally, one or more of:
(a) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, comprising a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(b) the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-0- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(c) the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16; wherein at least one of the polypeptide is a recombinant polypeptide; and producing the one or more steviol glycosides or glycosides of the steviol precursor thereby.
18. The method of claim 17, wherein the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group comprises at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T1 10P, 111 1V, A113C, 1115V, M1 19F, T120L, E121 P, V123A, I128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I 156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N211 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K311 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S411 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
19. The method of claim 17 or 18, wherein the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, an archaeal cell, or a bacterial cell.
20. The method of claim 17 or 18, wherein the recombinant host cell is a Saccharomyces cerevisiae cell.
21 . The method of claim 17 or 18, wherein the recombinant host cell is a Yarrowia lipolytica cell.
22. An in vitro method for producing one or more steviol glycosides or glycosides of a steviol precursor comprising adding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 11 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191 -195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-41 1 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4;
and, optionally, one or more of:
(a) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, comprising a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(b) the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-0- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(c) the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, comprising a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;
and a plant-derived or synthetic steviol, steviol precursors, and/or steviol glycosides to a reaction mixture;
wherein at least one of the polypeptide is a recombinant polypeptide; and producing the one or more steviol glycosides or glycosides of the steviol precursor thereby. The method of claim 22, wherein the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group comprises at least one amino acid substitution of SEQ ID NO:4 that is L15V, I16L, F18Y, L20A, F27M, I28L, F30L, G31S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T110P, 111 1V, A1 13C, 11 15V, M1 19F, T120L, E121 P, V123A, I128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I 156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N211 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K311 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S411 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
The method of claim 22 or 23, wherein the reaction mixture comprises:
(a) one or more steviol glycosides or glycosides of the steviol precursor;
(b) a UGT polypeptide;
(c) uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or
(d) reaction buffer and/or salts.
The method of any one of claims 10-24, wherein the one or more steviol glycosides or glycosides of the steviol precursor comprises 19-KMG, 13-SMG, steviol-1 ,2-Bioside, steviol-1 ,3-Bioside, 19-SMG, 1 ,2-Stevioside, RebG, rubusoside, RebA, RebB, RebC, RebD, RebE, RebF, RebM, RebQ, Rebl, dulcoside A, a mono-glycosylated ent- kaurenoic acid, a di-glycosylated enf-kaurenoic acid, a tri-glycosylated eni-kaurenoic acid, a mono-glycosylated enf-kaurenols, a di-glycosylated enf-kaurenol, a tri- glycosylated en/-kaurenol, a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and/or an isomer thereof.
26. A cell culture, comprising the recombinant host cell of any one of claims 1-9, the cell culture further comprising:
(a) the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, UDP-glucose, UDP- rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids;
wherein the one or more steviol glycosides or glycosides of the steviol precursor are present at a concentration of at least 1 mg/liter of the cell culture;
wherein the cell culture is enriched for the one or more steviol glycosides or glycosides of the steviol precursor relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
27. A cell lysate from the recombinant host cell of any one of claims 1 -9 grown in the cell culture, comprising:
(a) the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, UDP-glucose, UDP- rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base, YNB, and/or amino acids;
wherein the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell is present at a concentration of at least 1 mg/liter of the cell culture.
28. One or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell of any one of claims 1 -9;
wherein the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
29. One or more steviol glycosides or glycosides of the steviol precursor produced by the method of any one of claims 10-25;
wherein the one or more steviol glycosides or glycosides of the steviol precursor produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
30. A sweetener composition, comprising the one or more steviol glycosides or glycosides of the steviol precursor of claim 28 or 29.
31 . A food product, comprising the sweetener composition of claim 30.
32. A beverage or a beverage concentrate, comprising the sweetener composition of claim 30.
33. An isolated nucleic acid molecule encoding a polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86-88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191 -195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387- 391 , 396, 401 , 407-41 1 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4.
34. An isolated nucleic acid molecule encoding a polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having at least one amino acid substitution of SEQ ID NO:4 that is L15V, I 16L, F18Y, L20A, F27M, I28L, F30L, G31S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T110P, 11 11V, A1 13C, 11 15V, M1 19F, T120L, E121 P, V123A, I128K, E129Q, G135A, S136A, Q140N, A141S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N211 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S41 1 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
35. The nucleic acid of claim 33 or 34, wherein the nucleic acid is cDNA.
36. A polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having one or more amino acid substitutions of residues 15, 16, 18, 20, 27, 28, 30, 31 , 49, 51 , 67, 68, 73, 75, 79, 81 , 83, 84, 86 88, 90, 91 , 96, 99, 107, 1 10, 1 1 1 , 1 13, 1 15, 1 19-121 , 123, 128, 129, 135, 136, 140, 141 , 143, 146, 147, 156, 162, 166, 169, 173, 176, 179-181 , 183-189, 191 -195, 200, 204, 209, 21 1 , 212, 215, 221 , 222, 224, 232, 237, 247, 252, 255, 257, 259, 263, 265, 266, 269, 274, 280, 284, 285, 287, 292, 295-298, 300, 301 , 303, 310, 31 1 , 313, 315, 316, 320, 322, 325, 326, 328, 329, 332, 333, 335, 338, 341 , 346, 347, 357, 364, 370, 371 , 373, 375-377, 380, 385, 387-391 , 396, 401 , 407-411 , 415, 416, 419, 424, 426, 427, 434, 448, 449, 455, 456, or 458 of SEQ ID NO:4.
A polypeptide or a catalytically active portion thereof capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, wherein the encoded polypeptide or the catalytically active portion thereof comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 and having at least one amino acid substitution of SEQ ID NO:4 that is L15V, I 16L, F18Y, L20A, F27M, I28L, F30L, G31 S, G31A, T49I, N51 K, Q67E, A68T, C73F, E75D, M79A, E83D, E83K, S84A, L86I, E87D, T88R, K90W, Q91 E, S96T, D99E, E107S, T1 10P, 11 1 1V, A1 13C, 11 15V, M1 19F, T120L, E121 P, V123A, I128K, E129Q, G135A, S136A, Q140N, A141 S, V143A, S146N, L147I, I156L, E162T, V166L, F169L, Q173E, E176D, L179S, I180F, L181V, N183D, H184P, E185G, Q186S, I187Y, Q188P, S189A, W191 F, S192D, Q193M, M194V, L195V, A200S, Q204K, F209L, N21 1 H, S212T, K215E, 1221V, E222D, T224M, V232T, L237I, D247E, N252Y, N255S, Y257F, A259P, E263A, M265I, N266K, N266E, D269N, E274G, A280S, L284M, V285A, H287L, V292M, I295L, I295M, T296A, R297W, A298G, I300K, D301 N, D303N, 1310V, K31 1 R, K313S, E315Q, G316A, E320K, L322F, V325E, I326T, T328S, G329E, L332I, I333V, A335S, K338P, D341 E, E346P, E346K, S347A, F357W, I364L, V370M, V371 I, M373V, Q375L, F376W, S377T, T380S, L385F, D387E, E388D, I389V, L390W, G391 K, V396A, N401 K, G407E, N408E, L409I, A410E, S411 D, M415E, 1416V, E419G, I424E, R426K, K427E, D434E, N448K, D449N, S455A, E456K, or I458V.
The polypeptide or the catalytically active portion thereof of claim 36 or 37, wherein the polypeptide or the catalytically active portion thereof is a purified polypeptide or a catalytically active portion thereof.
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